Mycobacterial genes down-regulated during latency

ABSTRACT

A method is provided for identifying  mycobacterial  genes the expression of which is down-regulated during a stationary phase culture of  mycobacteria  under nutrient-starving conditions when compared with an exponential phase culture of  mycobacteria  under culture conditions that are not nutrient-starving and that support exponential growth of said  mycobacteria.  The described method optionally provides for identifying  mycobacterial  genes that are simultaneously down-regulated under low DOT conditions. The down-regulated genes of the present invention form the basis of nucleic acid vaccines, or provide targets to allow preparation of attenuated  mycobacteria  for vaccines against  mycobacterial  infections. Similarly, peptides encoded by said down-regulated genes are employed in vaccines. In a further embodiment, the identified genes/peptides provide the means for identifying the presence of a  mycobacterial  infection in a clinical sample by nucleic acid probe or antibody detection.

[0001] The present invention relates to a method of identifying a gene in mycobacteria the expression of which is down-regulated during mycobacterial latency, to the isolated peptide products, variants, derivatives or fragments thereof and to inhibitors thereof, to antibodies that bind to said peptides, variants, derivatives or fragments, to DNA and RNA vectors that express said polypeptide, variants, derivatives or fragments, to attenuated mycobacteria in which the activity of at least one of said genes has been modified, to vaccines against mycobacterial infections, and to methods of detecting the presence of a mycobacterial infection.

[0002] Many microorganisms are capable of forming intracellular infections. These include: Infections caused by species of Salmonella, Yersinia, Shigella, Campylobacter, Chlamydia and Mycobacteria. Some of these infections are exclusively intracellular, others contain both intracellular and extracellular components. However, it is the intracellular survival cycle of bacterial infection which is suspected as a main supportive factor for disease progression.

[0003] Generally, these microorganisms do not circulate freely in the body, for example, in the bloodstream, and are often not amenable to drug treatment regimes. Where drugs are available, this problem has been exacerbated by the development of multiple drug resistant microorganisms.

[0004] A number of factors have contributed to the problem of microbial resistance. One is the accumulation of mutations over time and the subsequent horizontal and vertical transfer of the mutated genes to other organisms. Thus, for a given pathogen, entire classes of antibiotics have been rendered inactive. A further factor has been the absence of a new class of antibiotics in recent years. The emergence of multiple drug-resistant pathogenic bacteria represents a serious threat to public health and new forms of therapy are urgently required.

[0005] For similar reasons, vaccine therapies have not proved effective against such intracellular microorganisms. Also, increased systemic concentration of antibiotics to improve bioavailability within cells may result in severe side effects.

[0006]Mycobacterium tuberculosis (TB) and closely related species make up a small group of mycobacteria known as the Mycobacterium tuberculosis complex (MTC). This group comprises five species M. tuberculosis, M. microti, M. bovis, M. caneti, and M. africanum which are the causative agent in the majority of tuberculosis (TB) cases throughout the world.

[0007]M. tuberculosis is responsible for more than three million deaths a year world-wide. Other mycobacteria are also pathogenic in man and animals, for example M. avium subsp. paratuberculosis which causes Johne's disease in ruminants, M. bovis which causes tuberculosis in cattle, M. avium and M. intracellulare which cause tuberculosis in immunocompromised patients (eg. AIDS patients, and bone marrow transplant patients) and M. leprae which causes leprosy in humans. Another important mycobacterial species is M. vaccae.

[0008]M. tuberculosis infects macrophage cells within the body. Soon after macrophage infection, most M. tuberculosis bacteria enter and replicate within cellular phagosome vesicles, where the bacteria are sequestered from host defences and extracellular factors.

[0009] It is the intracellular survival and multiplication or replication of bacterial infection which is suspected as a main supportive factor for mycobacterial disease progression.

[0010] A number of drug therapy regimens have been proposed for combatting M. tuberculosis infections, and currently combination therapy including the drug isoniazid has proved most effective. However, one problem with such treatment regimes is that they are long-term, and failure to complete such treatment can promote the development of multiple drug resistant microorganisms.

[0011] A further problem is that of providing an adequate bioavailability of the drug within the cells to be treated. Whilst it is possible to increase the systemic concentration of a drug (eg. by administering a higher dosage) this may result in severe side effects caused by the increased drug concentration.

[0012] The effectiveness of vaccine prevention against M. tuberculosis has varied widely. The current M. tuberculosis vaccine, BCG, is an attenuated strain of M. bovis. It is effective against severe complications of TB in children, but it varies greatly in its effectiveness in adults particularly across ethnic groups. BCG vaccination has been used to prevent tuberculous meningitis and helps prevent the spread of M. tuberculosis to extra-pulmonary sites, but does not prevent infection.

[0013] The limited efficacy of BCG and the global prevalence of TB has led to an international effort to generate new, more effective vaccines. The current paradigm is that protection will be mediated by the stimulation of a Th1 immune response.

[0014] BCG vaccination in man was given orally when originally introduced, but that route was discontinued because of loss of viable BCG during gastric passage and of frequent cervical adenopathy. In experimental animal species, aerosol or intra-tracheal delivery of BCG has been achieved without adverse effects, but has varied in efficacy from superior protection than parenteral inoculation in primates, mice and guinea pigs to no apparent advantage over the subcutaneous route in other studies.

[0015] Conventional mycobacterial vaccines, including BCG, protect against disease and not against infection. Ideally a new mycobacterial vaccine will impart sterile immunity, and a post-exposure vaccine capable of boosting the immune system to kill latent mycobacteria or prevent reactivation to active disease-causing microorganisms would also be valuable against latent infection.

[0016] There is therefore a need for an improved and/or alternative vaccine or therapeutic agent for combatting mycobacterial infections.

[0017] An additional major problem associated with the control of mycobacterial infections, especially M. tuberculosis infections, is the presence of a large reservoir of asymptomatic individuals infected with mycobacteria. Dormant mycobacteria are even more resistant to front-line drugs.

[0018] Infection with mycobacteria (eg. M. tuberculosis) rarely leads to active disease, and most individuals develop a latent infection which may persist for many years before reactivating to cause disease (Wayne, 1994). The current strategy for controlling such infection is early detection and treatment of patients with active disease. Whilst this is essential to avoid deaths and control transmission, it has no effect on eliminating the existing reservoir of infection or on preventing new cases of disease through reactivation.

[0019] Furthermore, conventional methods for the detection of a latent mycobacterial infection by skin testing may be compromised. For example, current TB detection methods based on tuberculin skin testing are compromised by BCG vaccination and by exposure to environmental mycobacteria.

[0020] Thus, new strategies are required for more effective diagnosis, treatment and prevention of mycobacterial latent infection.

[0021] To develop specific strategies for addressing latent mycobacterial infection it is necessary to elucidate the physiological, biochemical and molecular properties of these microorganisms.

[0022] However, at present, there is no suitable in vivo model for studying mycobacterial latent infection and such a model is unlikely to provide sufficient microbial material to enable detailed analysis of the physiological and molecular changes that occur.

[0023] In this respect, conventional mycobacterial culture systems for analysing gene and protein expression profiles have been based on simple, crude batch-type systems, such as those described in Sherman, D. R et al (2001) PNAS Vol. 98, No. 13, pp. 7534-7539; Imboden, P. (1998) Gene 213, pp. 107-117; Boon, C. et al (2001) J. Bacteriol. Vol. 183, No. 8, pp. 2672-2676; Cunningham, A. F et al (1998), J. Bacteriol. Vol. 180, No. 4, pp. 801-808; and Murugasu-Oei, B. et al (1999), Mol. Gen. Genet., Vol. 262, pp. 677-682. In these crude batch systems, mycobacterial growth follows a typical sigmoid growth curve involving an exponential growth phase and a stationary phase. The transition from exponential phase to stationary phase involves rapid and transient switches in terms of gene and protein expression, which switches are initiated by a complex set of undefined or poorly defined interactive stimuli as the mycobacteria become starved of essential nutrients.

[0024] In summary, studies to date have used either static cultures that allow tubercle bacilli to generate oxygen-depletion-gradients and enter a non-replicating persistent state in the sediment layer, or agitated sealed liquid cultures (Wayne and Lin, 1982; Cunningham and Spreadbury, 1998; Wayne and Hayes, 1996). Transition to a non-replicating persistent state in these models coincides with a shift-down to glyoxylate metabolism, resistance to isoniazid and rifampicin and susceptibility to the anaerobic bactericidal action of metronidazole (Wayne and Hayes, 1996).

[0025] Such studies are poorly defined and controlled, and experiments relying on self-generated oxygen-depletion gradients have yielded inconsistent results. Furthermore, these studies do not consider other physicochemical stimuli that may be important in vivo, and have been conducted over a short duration, in many cases 2-3 weeks.

[0026] In view of the above, there is a need for a defined and controlled model for studying mycobacterial (eg. TB) persistence, which simulates key features of the in vivo environment.

[0027] According to a first aspect of the invention there is provided an isolated mycobacterial peptide, or a fragment or derivative or variant thereof, wherein the peptide is encoded by a mycobacterial gene the expression of which gene is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving and which support exponential growth of said mycobacteria.

[0028] The following various embodiments (eg. preferred culture conditions, and sampling times) described for the first aspect of the present invention apply equally to the second and subsequent aspects of the present invention.

[0029] During infection, mycobacteria (eg. M. tuberculosis) encounter a dynamic host environment and modulate the expression of genes required for infection.

[0030] The selection conditions of the present invention permit identification of mycobacterial genes that are not required for maintenance of mycobacterial latency. Such selection conditions therefore permit the identification of genes that may be expressed in vivo during active mycobacterial infection, or during/following re-activation of a mycobacterium from a latent state. Active infection refers to an infectious state in which the mycobacteria demonstrate high metabolic activity and undergo cell division. Latency is discussed in more detail below.

[0031] The term latency is synonymous with persistence, and describes a reversible state of low metabolic activity in which mycobacterial cells can survive for extended periods without cell division. During latency (ie. latent infection), the clinical symptoms associated with a mycobacterial infection do not become manifest, and the host suffers no significant ill.

[0032] However, re-activation of latent mycobacteria may be induced by environmental stimuli, resulting in the development of an active mycobacterial infection with the associated clinical symptoms. The present inventors believe that stimuli such as an increase in nutrient availability, optionally together with an increase in the local dissolved oxygen concentration may induce re-activation.

[0033] For example, re-activation of latent mycobacteria may occur when previously limiting nutrients, and optionally oxygen, become available. This may happen, for example, following breakdown of a mycobacterial infection granuloma structure.

[0034] Studies of TB pathogenesis have established that tubercle bacilli reside in a number of different locations in the lung, for example lining the walls of cavitary lesions where the close proximity to bronchioles provides a source of air; within macrophages where oxygen availability may be more limited or within granulomas. Studies of granuloma structure, where the bacilli are encased by layers of macrophages and lymphocytes and may persist for prolonged periods, have led scientists to the conclusion that the environment within a granuloma is most likely to be anoxic. Therefore TB may encounter a range of atmospheric conditions from the point of transmission and inhalation through to long-term persistence in a granuloma.

[0035] The present inventors believe that mycobacteria such as M. tuberculosis must be able to adapt and respond to changes in nutrient, and optionally oxygen, availability in order to cause infection. The initial stages of infection, which include dissemination and phagocytosis, occur under aerobic conditions, and tubercle bacilli replicate rapidly in the aerobic environments of the cavitary lesion as well as within macrophages.

[0036] In vitro studies have demonstrated that mycobacteria such as M. tuberculosis can adapt and survive under nutrient- and oxygen-depleted conditions, and can grow over a range of oxygen tensions. Adaptation to carbon starvation, and optionally to a low dissolved oxygen tension, triggers transition to a non-replicating persistent state in vitro that may be analogous to latency in vivo.

[0037] Antigens repressed during latency and that have been identified according to the present invention may play an early and important role in the development of an effective immune response against replicating bacilli during the active stages of disease, and consequently represent good vaccine candidates. In addition, genes repressed under latency conditions are important therapeutic targets for preventing the establishment, spread and reactivation of disease. The identified genes are likely to be required for re-activation of latent mycobacteria, and replication of said re-activated bacilli. They are therefore key targets for the development of therapeutic agents and post-exposure vaccines to prevent re-activation of latent infection. Alternatively, said genes may be exploited in a treatment regime designed to trigger re-activation of latent bacilli. When re-activated, tubercle bacilli are more susceptible to treatment regimes. Thus, the antigens and genes of the present invention may form the basis for vaccines against pre- and post-infections by mycobacteria.

[0038] The term “nutrient-starving” in the context of the present invention means that the concentration of the primary carbon, and preferably the primary energy source, is insufficient to support optimal “exponential growth” of the mycobacteria. “Nutrient-starving” is a term that is generally associated with the stationary phase of a batch culture growth curve, or with the onset of late exponential/early stationary phase when the concentration of carbon is approaching depletion and starts to restrict the growth rate. Essential nutrients other than carbon (eg. nitrogen, iron, and oxygen) may also limit growth. Under such conditions the mycobacteria become metabolically stressed, rather than simply reduced in growth rate. By comparison, “nutrient limiting” is a term associated with continuous culture where growth is controlled/limited by the rate of addition of an essential nutrient to the culture system.

[0039] In more detail, exponential growth is that period of growth that is associated with a logarithmic increase in mycobacterial cell mass (also known as the “log” phase) when the bacteria are multiplying at their maximum specific growth rate for the prevailing culture conditions. During this period of growth the concentrations of essential nutrients diminish and those of end-products increase. However, once the primary carbon and/or primary energy source falls to below a critical level, it is no longer possible for all of the mycobacterial cells within the culture to obtain sufficient carbon and/or energy needed to support optimal cellular function and cell division. Once this occurs, exponential growth stops and the mycobacteria enter stationary phase. Growth may also cease due to the accumulation of inhibitory secondary metabolites, or as a result of pH changes.

[0040] Carbon starvation normally refers to a supply of exogenous carbon that is insufficient to enable the bacteria to grow/replicate. However, there may be other energy sources (eg. endogenous reserves, secondary metabolites) that are available to maintain essential-cellular functions and viability without supporting growth. Thus, carbon starvation is associated with a stationary phase condition in which the carbon source has become depleted and bacterial growth has substantially ceased.

[0041] During onset of stationary phase, DNA synthesis may continue for some time after net increase in cell mass has ceased, and the mycobacteria may divide to produce cells small in size and low in RNA content. Many proteins, including extracellular enzymes, may be synthesized during the stationary phase.

[0042] The onset of stationary phase vis-a-vis addition of a mycobacterial inoculum to the culture vessel will depend on a number of factors such as the particular mycobacterial species/strain, the composition of the culture media (eg. the particular primary carbon and energy source), and the physical culture parameters employed.

[0043] However, as a guide, the end of exponential phase and the onset of stationary phase generally corresponds to that point in the growth phase associated with the maximum number of viable counts of mycobacteria.

[0044] In use of the present invention, the exponential phase mycobacterial cells are harvested from the culture vessel at a point in the growth phase before the maximum number of total viable counts has been achieved. This point in the growth phase may be mimicked under continuous culture conditions employing a steady state growth rate approximating μ_(max) and providing a generation time of approximately 18-24 hours. In a preferred embodiment, the exponential phase mycobacterial cells are harvested when a value of between 2 and 0.5 (more preferably between 1 and 0.5) log units of viable counts per ml of culture medium less than the maximum number of viable counts per ml of culture medium has been achieved. Thus, the “exponential” phase cells are generally harvested during mid-log phase.

[0045] For example, if the maximum viable count value is 1*10¹⁰ per ml, then the “exponential” phase cells would be preferably harvested once a value of between 1*10⁸ and 1*10^(9.5) (more preferably between 1*10⁹ and 1*10^(9.5)) viable counts per ml has been achieved. In the case of M. tuberculosis, this would be approximately 3-10, preferably 4-7 days post-inoculation.

[0046] Similarly, in use of the present invention, the stationary phase mycobacterial cells are harvested from the culture vessel at a point in the growth phase after the maximum number of total viable counts has been achieved. This point in the growth phase may be mimicked under continuous culture conditions supporting a generation time of at least 3 days. In a preferred embodiment, the stationary phase mycobacterial cells are harvested when the viable counts per ml of culture medium has fallen to between 0.5 and 3 (more preferably between 1.5 and 2.5) log units less than the maximum number of viable counts per ml of culture medium. Thus, the “stationary” phase cells are generally harvested during mid- to late-stationary phase.

[0047] For example, if the maximum viable count value is 1*10¹⁰ per ml, then the stationary phase cells would be preferably harvested once the viable count number had fallen to a value of between 1*10^(9.5) and 1*10⁷ (more preferably between 1*10^(8.5) and 1*10^(7.5)) viable counts per ml. In the case of M. tuberculosis, this would be approximately day 25-60, preferably day 30-50, more preferably day 40-50, post-inoculation. For mycobacteria generally, the stationary phase cells are preferably harvested at least 25, preferably at least 30, more preferably at least 40 days post-inoculation. Longer post-inoculation harvesting times of at least 100 days, even at least 150 days may be employed. The above harvesting times apply to all aspects of the present invention.

[0048] The preferred culture method employed by the present invention is that of batch fermenter culture. In contrast to the crude prior art Wayne-type, simple batch culture systems, the batch fermenter system, of the present invention permits careful monitoring of growth culture parameters such as pH, temperature, available nutrients, and dissolved oxygen tension (DOT). In particular, temperature and DOT may be strictly controlled.

[0049] Thus, in use of the present method it is possible to ensure that the principal latency induction parameter employed is starvation of the primary carbon, and preferably the starvation of the primary carbon and energy source. In contrast, the crude prior art, simple batch systems simultaneously expose the cultured mycobacteria to a complex range of interactive environmental stimuli, which stimuli may obscure or modify the cellular effects associated with a single, principal stimulus in isolation.

[0050] Thus, the present invention substantially avoids the accidental induction or up-regulation of genes which are solely responsive to other environmental switches may be substantially prevented. Accordingly, false-positive identification of genes that are down-regulated under conditions unrelated to carbon starvation and/or energy limitation may be substantially avoided.

[0051] Suitable media for culturing mycobacteria are described in Wayne, L. G. (1994) [in Tuberculosis: Pathogenesis, Protection, and Control published by the American Society for Microbiology, pp. 73-83]. These include Middlebrook 7H9 Medium [see Barker, L. P., et al. (1998) Molec. Microbiol., vol. 29(5), pp. 1167-1177; and WO00/52139 in the name of the present Applicant].

[0052] In use of the method, the starting concentration of the primary carbon source (and preferably the primary energy source) is at least 0.5, preferably at least 1 gl⁻¹ of culture medium. Such concentrations are generally considered to be non nutrient-starving. Similarly, the onset of the stationary phase is generally associated with a primary carbon and energy source concentration of less than 0.5, preferably less than 0.2, and more preferably less than 0.1 gl⁻¹ of culture medium

[0053] In a preferred embodiment, the primary carbon and energy source is glycerol. The starting concentration of this component is at least 1, preferably 1-3, more preferably approximately 2 gl⁻¹ of culture medium. The onset of stationary phase is associated with a concentration of less than 0.2, preferably less than 0.1 gl⁻¹ of culture medium.

[0054] Other primary carbon and energy sources may be employed such as glucose, pyruvate, and fatty acids (eg. palmitate, and butyrate). These sources may be employed at substantially the same concentrations as for glycerol.

[0055] The pH of the culture medium is preferably maintained between pH 6 and 8, more preferably between pH 6.5 and 7.5, most preferably at about pH 6.9.

[0056] In one embodiment, the dissolved oxygen tension (DOT) is maintained throughout the culture process at at least 40% air saturation when measured at 37° C., more preferably between 50 and 70 % air saturation when measured at 37° C., most preferably at 50% air saturation when measured at 37° C.

[0057] The dissolved oxygen tension parameter is calculated by means of an oxygen electrode and conventional laboratory techniques at 37° C. Thus, 100% air saturation corresponds to a solution which is saturated with air, whereas 0% corresponds to a solution which has been thoroughly purged with an inert gas such as nitrogen. Calibration is performed under standard atmospheric pressure conditions and 37° C., and with conventional air comprising approximately 21% oxygen. Thus, the DOT values quoted in the present application concern DOT when measured at 37° C. and standard atmospheric pressure. A reference temperature and pressure is indicated, as DOT may vary with temperature and pressure.

[0058] In another embodiment of the present invention, latency may be induced by a combination of carbon and/or energy source starvation, and a low DOT.

[0059] In a preferred embodiment, the DOT is maintained at at least 40% air saturation, more preferably between 50 and 70% air saturation, until the mycobacterial culture has entered early-mid log phase. The DOT may be then lowered so as to become limiting, for example in increments over a 5 or 6 day period, and the culture maintained at a DOT of 0-10, preferably at a DOT of approximately 5% or less until the stationary phase cells are harvested.

[0060] The present inventors believe that the carbon and energy starvation, and optional low oxygen tension, induction conditions of the present invention are culture conditions that are conducive for a mycobacterium to express at least one gene that would be normally expressed in vivo during infection.

[0061] In use, it is preferred that those genes (ie. as represented by cDNAs in the detection assay) that are down-regulated by at least 1.5-fold under stationary phase, nutrient-starving conditions vis-a-vis exponential phase, non nutrient-starving conditions are selected. In more preferred embodiments, the corresponding down-regulation selection criterion is at least 2-fold, more preferably 3-fold, most preferably 4-fold. In further embodiments down-regulation levels of at least 10-fold, preferably 50-fold may be employed. The above down-regulation criteria apply to all aspects of the present invention.

[0062] The mycobacterium is selected from the species M. phlei, M. smegmatis, M. africanum, M. caneti, M. fortuitum, M. marinum, M. ulcerans, M. tuberculosis, M. bovis, M. microti, M. avium, M. paratuberculosis, M. leprae, M. lepraemurium, M. intracellulare, M. scrofulaceum, M. xenopi, M. genavense, M. kansasii, M. simiae, M. szulgai, M. haemophilum, M. asiaticum, M. malmoense, M. vaccae, M. caneti, and M. shimoidei. Of particular interest are members of the MTC, preferably M. tuberculosis.

[0063] The term peptide throughout this specification is synonymous with protein.

[0064] Use of mycobacterial peptide compositions according to the present invention provide excellent vaccine candidates for targeting mycobacteria in patients infected with mycobacteria.

[0065] The terms “isolated,” “substantially pure,” and “substantially homogenous” are used interchangeably to describe a peptide which has been separated from components which naturally accompany it. A peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide sequence. A substantially pure peptide will typically comprise about 60 to 90% w/w of a protein sample, more usually about 95%, and preferably will be over about 99% pure. Peptide purity or homogeneity may be indicated by, for example, polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel. Alternatively, higher resolution may be provided by using, for example, HPLC.

[0066] A peptide is considered to be isolated when it is separated from the contaminants which accompany it in its natural state. Thus, a peptide which is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.

[0067] The present invention provides peptides which may be purified from mycobacteria as well as from other types of cells transformed with recombinant nucleic acids encoding these peptides.

[0068] If desirable, the amino acid sequence of the proteins of the present invention may be determined by protein sequencing methods.

[0069] The terms “peptide”, “oligopeptide”, “polypeptide”, and “protein” are used interchangeably and do not refer to a specific length of the product. These terms embrace post-translational modifications such as glycosylation, acetylation, and phosphorylation.

[0070] The term “fragment” means a peptide having at least five, preferably at least ten, more preferably at least twenty, and most preferably at least thirty-five amino acid residues of the peptide which is the gene product of the down-regulated gene in question. The fragment preferably includes at least one epitope of the gene product in question.

[0071] The term “variant” means a peptide or peptide fragment having at least seventy, preferably at least eighty, more preferably at least ninety percent amino acid sequence homology with the peptide that is the gene product of the down-regulated gene in question. An example of a “variant” is a peptide or peptide fragment of a down-regulated gene which contains one or more analogs of an amino acid (eg. an unnatural amino acid), or a substituted linkage. The terms “homology” and “identity” are considered synonymous in this specification. In a further embodiment, a “variant” may be a mimic of the peptide or peptide fragment, which mimic reproduces at least one epitope of the peptide or peptide fragment. The mimic may be, for example, a nucleic acid mimic, preferably a DNA mimic.

[0072] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences may be compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequent coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percentage sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0073] Optimal alignment of sequences for comparison may be conducted, for example, by the local homology alignment algorithm of Smith and Waterman [Adv. Appl. Math. 2: 484 (1981)], by the algorithm of Needleman & Wunsch [J. Mol. Biol. 48: 443 (1970)] by the search for similarity method of Pearson & Lipman [Proc. Nat'l. Acad. Sci. USA 85:2444 (1988)], by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA—Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705), or by visual inspection [see Current Protocols in Molecular Biology, F. M. Ausbel et al, eds, Current Protocols, a joint venture between Greene Publishing Associates, In. And John Wiley & Sons, Inc. (1995 Supplement) Ausbubel].

[0074] Examples of algorithms suitable for determining percent sequence similarity are the BLAST and BLAST 2.0 algorithms [see Altschul (1990) J. Mol. Biol. 215: pp. 403-410; and “http://www.ncbi.nlm.nih.gov/” of the National Center for Biotechnology Information].

[0075] In a preferred homology comparison, the identity exists over a region of the sequences that is at least 10 amino acid, preferably at least 20 amino acid, more preferably at least 30 amino acid residues in length.

[0076] The term “derivative” means a protein comprising the peptide (or fragment, or variant thereof) which peptide is the gene product of the down-regulated gene in question. Thus, a derivative may include the peptide in question, and a further peptide sequence which may introduce one or more additional epitopes. The further peptide sequence should preferably not interfere with the basic folding and thus conformational structure of the peptide in question. Examples of a “derivative” are a fusion protein, a conjugate, and a graft. Thus, two or more peptides (or fragments, or variants) may be joined together to form a derivative. Alternatively, a peptide (or fragment, or variant) may be joined to an unrelated molecule (eg. a second, unrelated peptide). Derivatives may be chemically synthesized, but will be typically prepared by recombinant nucleic acid methods. Additional components such as lipid, and/or polysaccharide, and/or polyketide components may be included.

[0077] All of the molecules “fragment”, “variant” and “derivative” have a common antigenic cross-reactivity and/or substantially the same in vivo biological activity as the gene product of the down-regulated gene in question from which they are derived. For example, an antibody capable of binding to a fragment, variant or derivative would be also capable of binding to the gene product of the down-regulated gene in question. It is a preferred feature that the fragment, variant and derivative each possess the active site of the peptide which is the down-regulated peptide in question. Alternatively, all of the above embodiments of a peptide of the present invention share a common ability to induce a “recall response” of a T-lymphocyte which has been previously exposed to an antigenic component of a mycobacterial infection.

[0078] In a preferred embodiment, the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and 45.

[0079] According to a second aspect of the present invention there is provided a method of identifying a mycobacterial gene the expression of which is down-regulated during mycobacterial latency, said method comprising:

[0080] culturing a first mycobacterium under culture conditions that are nutrient-starving and which do not support exponential growth of the first mycobacterium;

[0081] culturing a second mycobacterium under culture conditions that are not nutrient-starving and which support exponential growth of the second mycobacterium;

[0082] obtaining first and second mRNA populations from said first and second mycobacteria respectively, wherein said first mRNA population is obtained from the first mycobacterium which has been harvested during stationary phase and wherein said second mRNA is obtained from the second mycobacterium which has been harvested during exponential phase growth;

[0083] preparing first and second cDNA populations from said first and second mRNA populations respectively, during which cDNA preparation a detectable label is introduced into the cDNA molecules of the first and second cDNA populations;

[0084] isolating corresponding first and second cDNA molecules from the first and second cDNA populations, respectively;

[0085] comparing relative amounts of label or corresponding signal emitted from the label present in the isolated first and second cDNA molecules;

[0086] identifying a greater amount of label or signal provided by the isolated second cDNA molecule than that provided by the isolated first cDNA molecule; and

[0087] identifying the first cDNA and the corresponding mycobacterial gene which is down-regulated during mycobacterial latency.

[0088] Reference to “down-regulated” embraces switched-off. A gene is switched-off when there is substantially no transcription of said gene.

[0089] Reference to gene throughout this specification embraces open reading frames (ORFs).

[0090] The term “corresponding first and second cDNA molecules from the first and second cDNA populations” refers to cDNAs having substantially the same nucleotide sequence. Thus, by isolating the cDNA copies relating to a given gene under each culture condition (ie. exponential phase, and stationary phase), it is possible to quantify the relative copy number of cDNA for that gene for each culture condition. Since each cDNA copy has been produced from an mRNA molecule, the cDNA copy number reflects the corresponding mRNA copy number for each culture condition, and thus it is possible to identify down-regulated genes.

[0091] In one embodiment, the first and second cDNA molecules are isolated from the corresponding first and second cDNA populations by hybridisation to an array containing immobilised DNA sequences which are representative of each known gene (or ORF) of a particular mycobacterial species' genome. Thus, a first cDNA may be considered “corresponding” to a second cDNA if both cDNAs hybridise to the same immobilised DNA sequence. Alternatively, representative DNA sequences from a particular mycobacterial strain, or from a number of different species and/or strains may be employed in the array.

[0092] In another embodiment, the first and second cDNAs are prepared by incorporation of a fluorescent label. The first and second cDNAs may incorporate labels which fluoresce at different wavelengths, thereby permitting dual fluorescence and simultaneous detection of two cDNA samples.

[0093] The type of label employed naturally determines how the output of the detection method is read. When using fluorescent labels, a confocal laser scanner is preferably employed.

[0094] According to one embodiment, fluorescently labelled cDNA sequences from stationary and exponential phase cultured systems were allowed to hybridise with a whole mycobacterial genome array. The first cDNA population was labelled with fluorescent label A, and the second cDNA population was labelled with fluorescent label B. The array was scanned at two different wavelengths corresponding to the excitable maxima of each dye and the intensity of the emitted light was recorded. Multiple arrays were then preferably prepared for each cDNA and a mean intensity value was calculated across the two cDNA populations for each spot with each dye, against which relative induction or up-regulation was quantified.

[0095] In addition to the above mRNA isolation and cDNA preparation and labelling, genomic DNA may be isolated from the first and second mycobacteria. Thus, in a preferred embodiment, labelled DNA is also prepared from the isolated DNA. The labelled DNA may be then included on each array as a control.

[0096] According to a third aspect of the present invention, there is provided an inhibitor of a mycobacterial peptide, wherein the peptide is encoded by a gene the expression of which is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving and that support exponential growth of said mycobacteria, and wherein the inhibitor is capable of preventing or inhibiting the mycobacterial peptide from exerting its native biological effect.

[0097] Such inhibitors may be employed to prevent the onset of, or to cause a break in the period of mycobacterial latency (ie. induce re-activation). In this respect, mycobacteria are more susceptible to treatment regimens when in a non-latent state, and the combined use of drugs to kill latent mycobacteria (eg. TB) would significantly reduce the incidence of mycobacteria by targeting the reservoir for new disease and would thereby help reduce the problem of emerging drug-resistant strains.

[0098] The inhibitor may be a peptide, carbohydrate, synthetic molecule, or an analogue thereof. Inhibition of the mycobacterial peptide may be effected at the nucleic acid level (ie. DNA, or RNA), or at the peptide level. Thus, the inhibitor may act directly on the peptide. Alternatively, the inhibitor may act indirectly on the peptide by, for example, causing inactivation of the down-regulated mycobacterial gene.

[0099] In preferred embodiments, the inhibitor is capable of inhibiting one or more of the following: endoglucanase, endo-1,4-beta-glucanase, carboxymethyl cellulase, inorganic phosphate transporter protein, transcriptional regulatory protein, 50S ribosomal protein L3, ribosomal protein S1, 30S ribosomal protein S4, uroporphyrin III C-methyltransferase, uroporphyrinogen III methylase, urogen III methylase, crystathionine gamma synthase, O-succinylhomoserine[thiol]-lyase, and zinc metalloprotease.

[0100] In a further embodiment, the inhibitor may be an antibiotic capable of targeting the down-regulated mycobacterial gene identifiable by the present invention, or the gene product thereof. The antibiotic is preferably specific for the gene and/or gene product.

[0101] In a further embodiment, the inhibitor may act on a gene or gene product the latter of which interacts with the down-regulated gene. Alternatively, the inhibitor may act on a gene or gene product thereof upon which the gene product of the down-regulated gene acts.

[0102] Inhibitors of the present invention may be prepared utilizing the sequence information provided herein. For example, this may be performed by overexpressing the peptide, purifying the peptide, and then performing X-ray crystallography on the purified peptide to obtain its molecular structure. Next, compounds are created which have similar molecular structures to all or portions of the peptide or its substrate. The compounds may be then combined with the peptide and attached thereto so as to block one or more of its biological activities.

[0103] Also included within the invention are isolated or recombinant polynucleotides that bind to the regions of the mycobacterial chromosome containing sequences that are associated with down-regulation under carbon starvation and optionally low oxygen tension (ie. virulence), including antisense and triplex-forming polynucleotides. As used herein, the term “binding” refers to an interaction or complexation between an oligonucleotide and a target nucleotide sequence, mediated through hydrogen bonding or other molecular forces. The term “binding” more specifically refers to two types of internucleotide binding mediated through base-base hydrogen bonding. The first type of binding is “Watson-Crick-type” binding interactions in which adenine-thymine (or adenine-uracil) and guanine-cytosine base-pairs are formed through hydrogen bonding between the bases. An example of this type of binding is the binding traditionally associated with the DNA double helix and in RNA-DNA hybrids; this type of binding is normally detected by hybridization procedures.

[0104] The second type of binding is “triplex binding”. In general, triplex binding refers to any type of base-base hydrogen bonding of a third polynucleotide strand with a duplex DNA (or DNA-RNA hybrid) that is already paired in a Watson-Crick manner.

[0105] In a preferred embodiment, the inhibitor may be an antisense nucleic acid sequence which is complementary to at least part of the inducible or up-regulatable gene.

[0106] The inhibitor, when in the form of a nucleic acid sequence, in use, comprises at least 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, and most preferably at least 50 nucleotides.

[0107] According to a fourth aspect of the invention, there is provided an antibody that binds to a peptide encoded by a gene, or to a fragment or variant or derivative of said peptide, the expression of which gene is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving.

[0108] The antibody preferably has specificity for the peptide in question, and following binding thereto may initiate coating of a mycobacterium expressing said peptide. Coating of the bacterium preferably leads to opsonization thereof. This, in turn, leads to the bacterium being destroyed. It is preferred that the antibody is specific for the mycobacterium (eg. species and/or strain) which is to be targeted.

[0109] In use, the antibody is preferably embodied in an isolated form.

[0110] Opsonization by antibodies may influence cellular entry and spread of mycobacteria in phagocytic and non-phagocytic cells by preventing or modulating receptor-mediated entry and replication in macrophages.

[0111] The peptides, fragments, variants or derivatives of the present invention may be used to produce antibodies, including polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal (eg. mouse, rabbit, goat, horse, etc.) is immunized with an immunogenic polypeptide. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to a desired mycobacterial epitope contains antibodies to other antigens, the polyclonal antibodies may be purified by immunoaffinity chromatography.

[0112] Alternatively, general methodology for making monoclonal antibodies by hybridomas involving, for example, preparation of immortal antibody-producing cell lines by cell fusion, or other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus may be employed.

[0113] The antibody employed in this aspect of the invention may belong to any antibody isotype family, or may be a derivative or mimic thereof. Reference to antibody throughout this specification embraces recombinantly produced antibody, and any part of an antibody which is capable of binding to a mycobacterial antigen.

[0114] In one embodiment the antibody belongs to the IgG, IgM or IgA isotype families.

[0115] In a preferred embodiment, the antibody belongs to, the IgA isotype family. Reference to the IgA isotype throughout this specification includes the secretory form of this antibody (ie. sigA). The secretory component (SC) of sigA may be added in vitro or in vivo. In the latter case, the use of a patient's natural SC labelling machinery may be employed.

[0116] In one embodiment, the antibody may be raised against a peptide from a member of the MTC, preferably against M. tuberculosis.

[0117] In a preferred embodiment, the antibody is capable of binding to a peptide selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, (or a fragment, variant, or derivative thereof). In a further embodiment, the antigen is an exposed component of a mycobacterial bacillus. In another embodiment, the antigen is a cell surface component of a mycobacterial bacillus.

[0118] The antibody of the present invention may be polyclonal, but is preferably monoclonal.

[0119] Without being bound by any theory, it is possible that following mycobacterial infection of a macrophage, the macrophage is killed and the bacilli are released. It is at this stage that the mycobacteria are considered to be most vulnerable to antibody attack. Thus, it is possible that the antibodies of the present invention act on released bacilli following macrophage death, and thereby exert a post-infection effect.

[0120] It is possible that the passive protection aspect (ie. delivery of antibodies) of the present invention is facilitated by enhanced accessibility of the antibodies of the present invention to antigens on mycobacterial bacilli. It is also possible that antibody binding may block macrophage infection by steric hindrance or disruption of its oligomeric structure. Thus, antibodies acting on mycobacterial bacilli released from killed, infected macrophages may interfere with the spread of re-infection to fresh macrophages. This hypothesis involves a synergistic action between antibodies and cytotoxic T cells, acting early after infection, eg. y{overscore (o)} and NK T cells, but could later involve also CD8 and CD4 cytotoxic T cells.

[0121] According to a fifth aspect of the invention, there is provided an attenuated mycobacterium in which a gene has been modified thereby rendering the mycobacterium substantially reduced in ability to enter a latent state, wherein said gene is a gene the expression of which is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving. The modification preferably inactivates the gene in question, and preferably renders the mycobacterium substantially non-pathogenic.

[0122] The term “modified” refers to any genetic manipulation such as a nucleic acid or nucleic acid sequence replacement, a deletion, or an insertion which renders the mycobacterium substantially reduced in ability to enter a latent state. In one embodiment the entire down-regulatable gene may be deleted.

[0123] In a preferred embodiment, gene to be modified has a wild-type coding sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46.

[0124] It will be appreciated that the above wild-type sequences may include minor variations depending on the Database employed. The term “wild-type” indicates that the sequence in question exists as a coding sequence in nature.

[0125] According to a sixth aspect of the invention, there is provided an attenuated microbial carrier, comprising a peptide encoded by a gene, or a fragment or variant or derivative of said peptide, the expression of which gene is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving.

[0126] In use, the peptide (or fragment, variant or derivative) is either at least partially exposed at the surface of the carrier, or the carrier becomes degraded in vivo so that at least part of the peptide (or fragment, variant or derivative) is otherwise exposed to a host's immune system.

[0127] In a preferred embodiment, the attenuated microbial carrier is attenuated salmonella, attenuated vaccinia virus, attenuated fowlpox virus, or attenuated M. bovis (eg. BCG strain).

[0128] In a preferred embodiment, the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45.

[0129] According to a seventh aspect of the invention, there is provided a DNA plasmid comprising a promoter, a polyadenylation signal, and a DNA sequence that encodes a gene or a fragment or variant of derivative of said gene, the expression of which gene is down-regulated during a stationary phase culture of mycobacteria under nutrient-starving culture conditions when compared with an exponential phase culture of mycobacteria under culture conditions that are not nutrient-starving, wherein the promoter and polyadenylation signal are operably linked to the DNA sequence. Reference to gene preferably means the peptide-coding sequence of the down-regulated gene.

[0130] The term DNA “fragment” used in this invention will usually comprise at least about 5 codons (15 nucleotides), more usually at least about 7 to 15 codons, and most preferably at least about 35 codons. This number of nucleotides is usually about the minimal length required for a successful probe that would hybridize specifically with such a sequence.

[0131] In preferred embodiments, the DNA “fragment” has a nucleotide length which is at least 50%, preferably at least 70%, and more preferably at least 80% that of the coding sequence of the corresponding down-regulated gene.

[0132] The term DNA “variant” means a DNA sequence which has substantial homology or substantial similarity to the coding sequence (or a fragment thereof) of a down-regulated gene. A nucleic acid or fragment thereof is substantially homologous (or “substantially similar”) to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95 to 98% of the nucleotide bases. Homology determination is performed as described supra for peptides.

[0133] Alternatively, a DNA “variant” is substantially homologous (or substantially similar) with the coding sequence (or a fragment thereof of a down-regulated gene when they are capable of hybridizing under selective hybridization conditions. Selectivity of hybridization exists when hybridization occurs which is substantially more selective than total lack of specificity. Typically, selective hybridization will occur when there is at least about 65% homology over a stretch of at least about 14 nucleotides, preferably at least about 70%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will often be over a stretch of at least about 17 nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

[0134] Nucleic acid hybridization will be affected by such conditions as salt concentration (eg. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of 30° C., typically in excess of 37° C. and preferably in excess of 45° C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. However, the combination of parameters is much more important than the measure of any single parameter. See, for example, Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.

[0135] The term DNA “derivative” means a DNA polynucleotide which comprises a DNA sequence (or a fragment, or variant thereof) corresponding to the coding sequence of the down-regulated gene and an additional DNA sequence which is not naturally associated with the DNA sequence corresponding to the coding sequence. The comments on peptide derivative supra also apply to DNA “derivative”. A “derivative” may, for example, include two or more coding sequences of a mycobacterial operon that is induced during nutrient starvation. Thus, depending on the presence of absence of a non-coding region between the coding sequences, the expression product/s of such as “derivative” may be a fusion protein, or separate peptide products encoded by the individual coding regions.

[0136] The above terms DNA “fragment”, “variant”, and “derivative” have in common with each other that the resulting peptide products have cross-reactive antigenic properties which are substantially the same as those of the corresponding wild-type peptide. Preferably all of the peptide products of the above DNA molecule embodiments of the present invention bind to an antibody which also binds to the wild-type peptide. Alternatively, all of the above peptide products are capable of inducing a “recall response” of a T lymphocyte which has been previously exposed to an antigenic component of a mycobacterial infection.

[0137] The promoter and polyadenylation signal are preferably selected so as to ensure that the gene is expressed in a eukaryotic cell. Strong promoters and polyadenylation signals are preferred.

[0138] In a related aspect, the present invention provides an isolated RNA molecule that is encoded by a DNA sequence of the present invention, or a fragment or variant or derivative of said DNA sequence.

[0139] An “isolated” RNA is an RNA which is substantially separated from other mycobacterial components that naturally accompany the sequences of interest, eg., ribosomes, polymerases, and other mycobacterial polynucleotides such as DNA and other chromosomal sequences.

[0140] The above RNA molecule may be introduced directly into a host cell as, for example, a component of a vaccine.

[0141] Alternatively the RNA molecule may be incorporated into an RNA vector prior to administration.

[0142] The polynucleotide sequences (DNA and RNA) of the present invention include a nucleic acid sequence that has been removed from its naturally occurring environment, and recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.

[0143] The term “recombinant” as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of a polynucleotide with which it is associated in nature; or (2) is linked to a polynucleotide other than that to which it is linked in nature; and (3) does not occur in nature. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, eg., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions.

[0144] In embodiments of the invention the polynucleotides may encode a peptide (or fragment, variant, or derivative) that is down-regulated under nutrient-starving conditions. A nucleic acid is said to “encode” a peptide if, in its native state or when manipulated, it can be transcribed and/or translated to produce the peptide (or fragment, variant or derivative thereof. The anti-sense strand of such a nucleic acid is also said to encode the peptide (or fragment, variant, or derivative).

[0145] Also contemplated within the invention are expression vectors comprising the polynucleotide of interest. Expression vectors generally are replicable polynucleotide constructs that encode a peptide operably linked to suitable transcriptional and translational regulatory elements. Examples of regulatory elements usually included in expression vectors are promoters, enhancers, ribosomal binding sites, and transcription and translation initiation and termination sequences. These regulatory elements are operably linked to the sequence to be translated. A nucleic acid sequence is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Generally, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. The regulatory elements employed in the expression vectors containing a polynucleotide encoding a virulence factor are functional in the host cell used for expression.

[0146] The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.

[0147] The polynucleotides of the present invention may also be produced by chemical synthesis, eg. by the phosphoramidite method or the triester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0148] DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system recognized by the host, including the intended DNA fragment encoding the desired peptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide encoding segment. Expression vectors may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Secretion signals from polypeptides secreted from the host cell of choice may also be included where appropriate, thus allowing the protein to cross and/or lodge in cell membranes, and thus attain its functional topology or be secreted from the cell.

[0149] Appropriate promoter and other necessary vector sequences are selected so as to be functional in the host, and may, when appropriate, include those naturally associated with mycobacterial genes. Promoters such as the trp, lac and phage promoters, tRNA promoters and glycolytic enzyme promoters may be used in prokaryotic hosts. Useful yeast promoters include the promoter regions for metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, enzymes responsible for maltose and galactose utilization, and others.

[0150] Appropriate non-native mammalian promoters may include the early and late promoters from SV40 or promoters derived from murine moloney leukemia virus, mouse mammary tumour virus, avian sarcoma viruses, adenovirus II, bovine papilloma virus or polyoma. In addition, the construct may be joined to an amplifiable gene (eg. DHFR) so that multiple copies of the gene may be made.

[0151] While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.

[0152] Expression and cloning vectors may contain a selectable marker, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. The presence of this gene ensures the growth of only those host cells which express the inserts. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, eg. ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media, e.g. the gene encoding D-alanine racemase for Bacilli. The choice of appropriate selectable marker will depend on the host cell.

[0153] The vectors containing the nucleic acids of interest can be transcribed in vitro and the resulting RNA introduced into the host cell (eg. by injection), or the vectors can be introduced directly into host cells by methods which vary depending on the type of cellular host, including electroporation; transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; infection (where the vector is an infectious agent, such as a retroviral genome). The cells into which have been introduced nucleic acids described above are meant to also include the progeny of such cells.

[0154] Large quantities of the nucleic acids and peptides of the present invention may be prepared by expressing the nucleic acids or portions thereof in vectors or other expression vehicles in compatible prokaryotic or eukaryotic host cells. The most commonly used prokaryotic hosts are strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas may also be used.

[0155] Mammalian or other eukaryotic host cells, such as those of yeast, filamentous fungi, plant, insect, amphibian or avian species, may also be useful for production of the proteins of the present invention. Propagation of mammalian cells in culture is per se well known. Examples of commonly used mammalian host cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines, although other cell lines may be appropriate, e.g., to provide higher expression, desirable glycosylation patterns.

[0156] Clones are selected by using markers depending on the mode of the vector construction. The marker may be on the same or a different DNA molecule, preferably the same DNA molecule. The transformant may be screened or, preferably, selected by any of the means well known in the art, e.g., by resistance to such antibiotics as ampicillin, tetracycline.

[0157] The polynucleotides of the invention may be inserted into the host cell by any means known in the art, including for example, transformation, transduction, and electroporation. As used herein, “recombinant host cells”, “host cells”, “cells”, “cell lines”, “cell cultures”, and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellular entities refer to cells which can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny of the original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. “Transformation”, as used herein, refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f-mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

[0158] In one embodiment, a DNA plasmid or RNA vector may encode a component of the immune system that is specific to an immune response following challenge with a peptide, wherein said peptide is encoded by a mycobacterial gene that is down-regulated under nutrient-starving culture conditions.

[0159] An example of such a component is an antibody to the peptide product of the down-regulated gene. Thus, in one embodiment, the nucleic acid sequence (eg. DNA plasmid or RNA vector) encodes the antibody in question.

[0160] In the context of the present invention, the term plasmid is equivalent to vector. Thus, a plasmid may be either linear or circularised nucleic acid.

[0161] An eighth aspect provides use of said aforementioned peptide or fragment or variant or derivative thereof, inhibitor, antibody, attenuated mycobacterium, attenuated microbial carrier, DNA sequence corresponding to the coding sequence of a mycobacterial gene that is down-regulated under nutrient-starving conditions or a fragment or variant or derivative of said DNA sequence, DNA plasmid comprising said DNA sequence or said fragment or variant or derivative, RNA sequence encoded by said DNA sequence or said fragment or variant or derivative, and/or RNA vector comprising said RNA sequence, in the manufacture of a medicament for treating or preventing a mycobacterial infection.

[0162] The term “preventing” includes reducing the severity/intensity of, or initiation of, a mycobacterial infection.

[0163] The term “treating” includes post-infection therapy and amelioration of a mycobacterial infection. In a related aspect, there is provided a method of treating or preventing a mycobacterial infection, comprising administration to a subject of a medicament selected from the group consisting of said aforementioned peptide or fragment or variant or derivative thereof, inhibitor, antibody, attenuated mycobacterium, attenuated microbial carrier, DNA sequence corresponding to the coding sequence of a mycobacterial gene that is down-regulated under nutrient-starving conditions or a fragment or variant or derivative of said DNA sequence, DNA plasmid comprising said DNA sequence or said fragment or variant or derivative, RNA sequence encoded by said DNA sequence or said fragment or variant or derivative, and/or RNA vector comprising said RNA sequence.

[0164] The immunogenicity of the epitopes of the peptides of the invention may be enhanced by preparing them in mammalian or yeast systems fused with or assembled with particle-forming proteins such as, for example, that associated with hepatitis B surface antigen. Vaccines may be prepared from one or more immunogenic peptides of the present invention.

[0165] Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmito yl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.

[0166] The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.

[0167] The peptides may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0168] The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 5 micrograms to 250 micrograms of antigen per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be peculiar to each subject.

[0169] The vaccine may be given in a single dose schedule, or preferably in a multiple dose schedule. A multiple dose schedule is one in which a primary course of vaccination may be with 1-10 separate doses, followed by other doses given at subsequent time intervals required to maintain and or re-enforce the immune response, for example, at 14 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also, at least in part, be determined by the need of the individual and be dependent upon the judgment of the practitioner.

[0170] In addition, the vaccine containing the immunogenic mycobacterial antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins, as well as antibiotics.

[0171] The medicament may be administered by conventional routes, eg. intravenous, intraperitoneal, intranasal routes.

[0172] The outcome of administering antibody-containing compositions may depend on the efficiency of transmission of antibodies to the site of infection. In the case of a mycobacterial respiratory infection (eg. a M. tuberculosis infection), this may be facilitated by efficient transmission of antibodies to the lungs.

[0173] In one embodiment the medicament may be administered intranasally (i.n.). This mode of delivery corresponds to the route of delivery of a M. tuberculosis infection and, in the case of antibody delivery, ensures that antibodies are present at the site of infection to combat the bacterium before it becomes intracellular and also during the period when it spreads between cells.

[0174] An intranasal composition may be administered in droplet form having approximate diameters in the range of 100-5000 μm, preferably 500-4000 μm, more preferably 1000-3000 μm. Alternatively, in terms of volume, the droplets would be in the approximate range of 0.001-100 μl, preferably 0.1-50 μl, more preferably 1.0-25 μl.

[0175] Intranasal administration may be achieved by way of applying nasal droplets or via a nasal spray.

[0176] In the case of nasal droplets, the droplets may typically have a diameter of approximately 1000-3000 μm and/or a volume of 1-25 μl.

[0177] In the case of a nasal spray, the droplets may typically have a diameter of approximately 100-1000 μm and/or a volume of 0.001-1 μl.

[0178] It is possible that, following i.n. delivery of antibodies, their passage to the lungs is facilitated by a reverse flow of mucosal secretions, although mucociliary action in the respiratory tract is thought to take particles within the mucus out of the lungs. The relatively long persistence in the lungs' lavage, fast clearance from the bile and lack of transport to the saliva of some antibodies suggest the role of mucosal site specific mechanisms.

[0179] In a different embodiment, the medicament may be delivered in an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution.

[0180] The size of aerosol particles is one factor relevant to the delivery capability of an aerosol. Thus, smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target a particular section of the respiratory airway, for example the alveoli.

[0181] The aerosol particles may be delivered by way of a nebulizer or nasal spray.

[0182] In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0.1-50 μm, preferably 1-25 μm, more preferably 1-5 μm.

[0183] The aerosol formulation of the medicament of the present invention may optionally contain a propellant and/or surfactant.

[0184] By controlling the size of the droplets which are to be administered to a patient to within the defined range of the present invention, it is possible to avoid/minimise inadvertent antigen delivery to the alveoli and thus avoid alveoli-associated pathological problems such as inflammation and fibrotic scarring of the lungs.

[0185] I.n. vaccination engages both T and B cell mediated effector mechanisms in nasal and bronchus associated mucosal tissues, which differ from other mucosae-associated lymphoid tissues.

[0186] The protective mechanisms invoked by the intranasal route of administration may include: the activation of T lymphocytes with preferential lung homing; upregulation of co-stimulatory molecules, eg. B7.2; and/or activation of macrophages or secretory IgA antibodies.

[0187] Intranasal delivery of antigens may facilitate a mucosal antibody response is invoked which is favoured by a shift in the T cell response toward the Th2 phenotype which helps antibody production. A mucosal response is characterised by enhanced IgA production, and a Th2 response is characterised by enhanced IL-4 production.

[0188] Intranasal delivery of mycobacterial antigens allows targeting of the antigens to submucosal B cells of the respiratory system. These B cells are the major local IgA-producing cells in mammals and intranasal delivery facilitates a rapid increase in IgA production by these cells against the mycobacterial antigens.

[0189] In one embodiment administration of the medicament comprising a mycobacterial antigen stimulates IgA antibody production, and the IgA antibody binds to the mycobacterial antigen. In another embodiment, a mucosal and/or Th2 immune response is stimulated.

[0190] In another embodiment monoclonal antibodies, in particular, may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an “internal image” of the antigen of the infectious agent against which protection is desired. These anti-idiotype antibodies may also be useful for treatment, vaccination and/or diagnosis of mycobacterial infections.

[0191] According to a ninety aspect of the present invention, the peptides (including fragments, variants, and derivatives thereof) of the present invention and antibodies that bind thereto are useful in immunoassays to detect the presence of antibodies to mycobacteria, or the presence of the virulence associated antigens in biological samples. Design of the immunoassays maybe subject to a great deal of variation, and many formats are known in the art. The immunoassay may utilize at least one epitope derived from a peptide of the present invention. In one embodiment, the immunoassay uses a combination of such epitopes. These epitopes may be derived from the same or from different bacterial peptides, and may be in separate recombinant or natural peptide or together in the same recombinant peptide.

[0192] An immunoassay may use, for example, a monoclonal antibody directed towards a virulence associated peptide epitope(s), a combination of monoclonal antibodies directed towards epitopes of one mycobacterial antigen, monoclonal antibodies directed towards epitopes of different mycobacterial antigens, polyclonal antibodies directed towards the same antigen, or polyclonal antibodies directed towards different antigens. Protocols may be based, for example, upon competition, or direct reaction, or sandwich type assays. Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labelled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the probe are also known; examples of which are assays which utilize biotin and avidin, and enzyme-labelled and mediated immunoassays, such as ELISA assays.

[0193] Typically, an immunoassay for an antibody(s) to a peptide, will involve selecting and preparing the test sample suspected of containing the antibodies, such as a biological sample, then incubating it with an antigenic (ie. epitope-containing) peptide(s) under conditions that allow antigen-antibody complexes to form, and then detecting the formation of such complexes. The immunoassay may be of a standard or competitive type.

[0194] The peptide is typically bound to a solid support to facilitate separation of the sample from the peptide after incubation. Examples of solid supports that can be used are nitrocellulose (eg. in membrane or microtiter well form), polyvinyl chloride (eg. in sheets or microtiter wells), polystyrene latex (eg. in beads or microtiter plates, polyvinylidine fluoride (known as Immulon), diazotized paper, nylon membranes, activated beads, and Protein A beads. For example, Dynatech Immulon microliter plates or 60 mm diameter polystyrene beads (Precision Plastic Ball) may be used. The solid support containing the antigenic peptide is typically washed after separating it from the test sample, and prior to detection of bound antibodies.

[0195] Complexes formed comprising antibody (or, in the case of competitive assays, the amount of competing antibody) are detected by any of a number of known techniques, depending on the format. For example, unlabelled antibodies in the complex may be detected using a conjugate of antixenogeneic Ig complexed with a label (eg. an enzyme label).

[0196] In immunoassays where the peptides are the analyte, the test sample, typically a biological sample, is incubated with antibodies directed against the peptide under conditions that allow the formation of antigen-antibody complexes. It may be desirable to treat the biological sample to release putative bacterial components prior to testing. Various formats can be employed. For example, a “sandwich assay” may be employed, where antibody bound to a solid support is incubated with the test sample; washed; incubated with a second, labelled antibody to the analyte, and the support is washed again. Analyte is detected by determining if the second antibody is bound to the support. In a competitive format, a test sample is usually incubated with antibody and a labelled, competing antigen is also incubated, either sequentially or simultaneously.

[0197] Also included as an embodiment of the invention is an immunoassay kit comprised of one or more peptides of the invention, or one or more antibodies to said peptides, and a buffer, packaged in suitable containers.

[0198] As used herein, a “biological sample” refers to a sample of tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumours, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).

[0199] In a related diagnostic assay, the present invention provides nucleic acid probes for detecting a mycobacterial infection.

[0200] Using the polynucleotides of the present invention as a basis, oligomers of approximately 8 nucleotides or more can be prepared, either by excision from recombinant polynucleotides or synthetically, which hybridize with the mycobacterial sequences, and are useful in identification of mycobacteria. The probes are a length which allows the detection of the down-regulated sequences by hybridization. While 6-8 nucleotides may be a workable length, sequences of 10-12 nucleotides are preferred, and at least about 20 nucleotides appears optimal. These probes can be prepared using routine methods, including automated oligonucleotide synthetic methods. For use as probes, complete complementarity is desirable, though it may be unnecessary as the length of the fragment is increased.

[0201] For use of such probes as diagnostics, the biological sample to be analyzed, such as blood or serum, may be treated, if desired, to extract the nucleic-acids contained therein. The resulting nucleic acid from the sample may be subjected to gel electrophoresis or other size separation techniques; alternatively, the nucleic acid sample may be dot blotted without size separation. The probes are usually labeled. Suitable labels, and methods for labeling probes are known in the art, and include, for example, radioactive labels incorporated by nick translation or kinasing, biotin, fluorescent probes, and chemiluminescent probes. The nucleic acids extracted from the sample are then treated with the labeled probe under hybridization conditions of suitable stringencies.

[0202] The probes may be made completely complementary to the virulence encoding polynucleotide. Therefore, usually high stringency conditions are desirable in order to prevent false positives. The stringency of hybridization is determined by a number of factors during hybridization and during the washing procedure, including temperature, ionic strength, length of time, and concentration of formamide.

[0203] It may be desirable to use amplification techniques in hybridization assays. Such techniques are known in the art and include, for example, the polymerase chain reaction (PCR) technique.

[0204] The probes may be packaged into diagnostic kits. Diagnostic kits include the probe DNA, which may be labeled; alternatively, the probe DNA may be unlabeled and the ingredients for labeling may be included in the kit in separate containers. The kit may also contain other suitably packaged reagents and materials needed for the particular hybridization protocol, for example, standards, as well as instructions for conducting the test.

[0205] In a preferred embodiment, a peptide (or fragment or variant or derivative) of the present invention is used in a diagnostic assay to detect the presence of a T-lymphocyte, which T-lymphocyte has been previously exposed to an antigenic component of a mycobacterial infection in a patient.

[0206] In more detail, a T-lymphocyte which has been previously exposed to a particular antigen will be activated on subsequent challenge by the same antigen. This activation provides a means for identifying a positive diagnosis of mycobacterial infection. In contrast, the same activation is not achieved by a T-lymphocyte which has not been previously exposed to the particular antigen.

[0207] The above “activation” of a T-lymphocyte is sometimes referred to as a “recall response” and may be measured, for example, by determining the release of interferon (eg. IFN-Y) from the activated T-lymphocyte. Thus, the presence of a mycobacterial infection in a patient may be determined by the release of a minimum concentration of interferon from a T-lymphocyte after a defined time period following in vitro challenge of the T-lymphocyte with a peptide (or fragment or variant or derivative) of the present invention. In use, a biological sample containing T-lymphocytes is taken from a patient, and then challenged with a peptide (or fragment, variant, or derivative thereof) of the present invention.

[0208] The above T-lymphocyte diagnostic assay may include an antigen presenting cell (APC) expressing at least one major histocompatibility complex (MHC) class II molecule expressed by the patient in question. The APC may be inherently provided in the biological sample, or may be added exogenously. In one embodiment, the T-lymphocyte is a CD4 T-lymphocyte.

[0209] Brief mention is now made to the Figures of the present application, in which:

[0210]FIG. 1 illustrates the viable counts for M. tuberculosis during culture under batch fermentation conditions at a DOT of 50% air saturation (37° C.);

[0211]FIG. 2 illustrates the concentration of glycerol (as the primary carbon and energy source) during culture of M. tuberculosis under batch fermentation conditions at the separate DOT concentrations of 0.5% and 50% air saturation (37° C.);

[0212]FIG. 3 illustrates the DOT within the medium of the mycobacterial culture described in Example 18; and

[0213]FIG. 4 illustrates the viable counts for M. tuberculosis during the batch fermentation conditions of Example 18 (ie. carbon-starvation, and oxygen limiting conditions).

[0214] Two alternative mycobacterial culture methods have been employed to study genes which are down-regulated during mycobacterial latency. The first method is described in Examples 1-8, whereas the second method is described in Example 18.

EXAMPLE 1 In vitro Model of mycobacterial Persistence Under Aerobic, Nutrient-Starved Conditions

[0215] Materials and Methods

[0216] Strain

[0217] Studies were performed with M. tuberculosis strain H37Rv (NCTC cat. no. 7416)—a representative strain of M. tuberculosis. Stock cultures were grown on Middlebrook 7H10+OADC for 3 weeks at 37±2° C.

[0218] Culture Medium

[0219] Persistence cultures were established in Middlebrook 7H9 medium supplemented with Middlebrook ADC enrichment, 0.2% Tween 80 and 0.2% glycerol (Table 1). The medium was prepared with high quality waterfrom a Millipore water purification system and filter sterilised by passage through a 0.1 μm pore size cellulose acetate membrane filter capsule (Sartorius Ltd). The pH was adjusted to 6.6 with concentrated hydrochloric acid.

[0220] Middlebrook 7H10+OADC agar was used to prepare inoculum cultures, enumerate the number of culturable bacteria in samples, and to assess culture purity.

[0221] Culture System

[0222] We previously developed a process for the culture of mycobacteria under controlled and defined conditions—Patent Application No. PCT/GB00/00760 (WO00/52139). We used this culture system operated as a batch fermenter for the following studies of mycobacterial persistence.

[0223] Culture experiments were performed in a one litre glass vessel operated at a working volume of 750 ml. The culture was agitated by a magnetic bar placed in the culture vessel coupled to a magnetic stirrer positioned beneath the vessel. Culture conditions were continuously monitored by an Anglicon Microlab Fermentation System (Brighton Systems, Newhaven), linked to sensor probes inserted into the culture through sealed ports in the top plate. The oxygen concentration was monitored with a galvanic oxygen electrode (Uniprobe, Cardiff) and was controlled through sparging the culture with a mixture of air and oxygen free-nitrogen. Temperature was monitored by an Anglicon temperature probe, and maintained by a heating pad positioned beneath the culture vessel. Culture pH was measured using an Ingold pH electrode (Mettler-Toledo, Leicester).

[0224] Inoculation and Culture

[0225] The vessel was filled with 750 ml of sterile culture medium and parameters were allowed to stabilise at 37° C.∓2° C., pH 6.9∓0.3 and a dissolved oxygen tension of approximately 70% air saturation. A dense inoculum suspension was prepared by resuspending Middlebrook agar cultures; grown at 37° C.∓2° C. for 3 weeks, in sterile deionised water. The inoculum was aseptically transferred to the culture vessel, to provide an initial culture turbidity of approximately 0.25 at 540 nm.

[0226] The culture were maintained at 37° C. with an agitation rate of 500 to 750 rpm. The dissolved oxygen tension was maintained between 50-70% air saturation with the aid of culture sparging. The initial culture pH was set at approximately 6.7 and was monitored throughout the experiment. The culture was maintained for 50 days and samples were removed regularly to monitor growth and survival, nutrient utilisation and gene expression.

[0227] Growth and Survival

[0228] Bacterial growth and survival was assessed by determining the number of viable cells in the culture system at specific time points. This was achieved by preparing a decimal dilution series of the sample in sterile water and plating 100 μl aliquots onto Middlebrook 7H10+OADC plates. The plates were incubated at 37° C. for up to 4 weeks before enumerating the number of colonies formed.

[0229] Nutrient Utilisation

[0230] Glycerol is the primary carbon and energy source present in Middlebrook 7H9 medium with ADC, 0.2% Tween and 0.2% Glycerol. The rate at which glycerol was utilised was determined using the Glycerol Determination Kit Cat. No. 148 270 Boehringer Mannheim.

[0231] Microarray Experiments

[0232] RNA was extracted from culture samples collected at different time points during the experiment. A fluorescently-labelled cDNA was then transcribed from each sample of RNA. The cDNA was labelled by the incorporation of either Cy3 or Cy5 labelled dCTP (Dyes are supplied by Amersham Pharmacia Biotech).

[0233] Whole M. tuberculosis genome arrays were prepared from M. tuberculosis genomic DNA using ORF-specific primers. PCR products corresponding to each ORF were spotted in a grid onto a standard glass microscope slide using a BioRobotics microgrid robot (MWG Biotech) at a resolution of >4000 spots/cm².

[0234] In each microarray experiment a whole genome array was hybridised with labelled cDNA from one culture sample (Test sample). Each array was also hybridised with control DNA incorporating a different Cy dye and prepared from DNA extracted from M. tuberculosis strain H37Rv (control sample).

[0235] Each array was scanned at two different wavelengths corresponding to the excitation maxima of each dye and the intensity of the emitted light was recorded. The ration of the intensity values for the test and control samples was determined for each array. The slides were scanned using an Affymetrix 428 scanner. The raw data was initially analysed by ImaGene software. The scanned images were then transferred to another software package known as GeneSpring to analyse the expression of each gene.

[0236] Results

[0237] After inoculation the culture entered exponential growth and continued to grow exponentially until 10 days after inoculation (see FIG. 1). Cessation of exponential growth coincided with depletion of the primary carbon and energy source—glycerol (see FIG. 2). As the culture entered stationary phase, viability started to decline and continued to decline steadily over the duration of the study. After 40 days in stationary phase, approximately 1% of the culture was still culturable on Middlebrook agar.

[0238] The gene expression profiles for samples collect at day 5 and day 50 were compared. Three arrays were prepared for each sample and the test data were normalised against the control data on each chip. The normalised data for each set of arrays were then averaged and the two sets of data were compared. Those genes that were expressed at least 1.5-fold less at day 50 relative to day 5 were selected. SEQ IDs 1-20 identified by this method are listed in Table 2, together with assigned biological functions for each of the encoded peptide.

[0239] The coding sequences (nucleic acid sequences are given from the transcription start site to the stop codon) for these genes and their corresponding amino acid sequences are listed in the accompanying sequence listing, in which the first SEQ lD NO is the amino acid sequence and the second SEQ ID NO is the corresponding nucleic acid coding sequence. TABLE 1 liquid medium formulation for persistence cultures - Middlebrook 7H9 medium supplemented with ADC, 0.2% Tween 80 and 0.2% Glycerol Composition per liter Na₂HPO₄ 2.5 g KH₂PO₄ 1.0 g Monosodium glutamate 0.5 g (NH₄)₂SO₄ 0.5 g Sodium citrate 0.1 g MgSO₄.7H₂O 0.05 g Ferric ammonium citrate 0.04 g CuSO₄.5H₂O 1.0 mg Pyridoxine 1.0 mg ZnSO₄.7H₂O 1.0 mg Biotin 0.5 mg CaCl₂.2H₂O 0.5 mg Middlebrook ADC enrichment 100 ml Glycerol 2.0 ml Tween 80 2.0 ml Middlebrook ADC enrichment - per 100 ml Bovine serum albumin 5.0 g Glucose 2.0 g Catalase 3.0 mg

[0240] TABLE 2 Genes down-regulated during survival under carbon starvation conditions. Fold- Gene down Assigned function SEQ ID NO: Rv0062; 6.9605 Cellulase CELA1 (endoglucanase) (endo-1,4- 1;2 celA (1F8) beta-glucanase) (Fl-CMASE) (carboxymethyl cellulase) Rv0545c; 5.0523 Low-affinity inorganic phosphate 3;4 pitA (4N16) transporter integral membrane protein PITA Rv0653c; 6.2012 Transcriptional regulatory protein 5;6 (4P23) Rv0701; 5.4226 50S ribosomal protein L3 RPLC 7;8 rplC (3N15) Rv1630; 5.815 Ribosomal protein S1 RpsA  9;10 rpsA (5E24) Rv1887; 5.8208 11;12 (7D11) Rv2144c; 6.0519 Transmembrane protein 13;14 (9G15) Rv3284; 5.4896 15;16 (4G3) Rv3458c; 5.432 30S ribosomal protein S4 RPSD 17;18 rpsD (4D18) Rv3478; 8.7897 PE family protein 19;20 PPE (4H19)

EXAMPLE 2 RNA Extraction from M. tuberculosis for Microarray Analysis

[0241] Materials and Methods

[0242] Trizol (Life Technologies) formulation of phenol and guanidine thiocyanate.

[0243] GTC lysis solution containing: 5 M guanidine thiocyanate, 0.5% N-lauryl sarcosine, 25 mM tri-sodium citrate, 0.1 M 2-mercaptoethanol, and 0.5% Tween 80.

[0244] Chloroform

[0245] Isopropanol

[0246] 3M sodium acetate

[0247] 70% Ethanol

[0248] microfuge

[0249] ribolyser

[0250] Sterile plasticware-Falcon tubes, screw capped eppendorfs, gilson tips—all RNase free

[0251] Glassware—baked at 160° C. for at least 16 hours

[0252] Method

[0253] Steps performed at Containment level 3; within a Class III microbiological safety cabinet.

[0254] Remove 10 or 20 ml of culture (10⁹/ml) and immediately add this to 4 volumes of GTC lysis buffer in a plastic specimen pot. Seal the pot tightly.

[0255] Incubate the cells in GTC lysis buffer for 1 hour at room temperature. Surface decontaminate the plastic pot with 5% Hycolin for 5 minutes. Transfer the sample to the pass box and place it into a plastic carry tin with a sealable lid. Close the container securely and transport it to a non-toxic cabinet CL3 cabinet.

[0256] Equally distribute the lysis mixture between Falcon tubes. Place these tubes into centrifuge buckets and seal the buckets tightly. Surface-decontaminate the buckets for 5 minutes with 5% Hycolin. Then transferthem tothe centrifuge (Baird and Tatlock Mark IV refrigerated bench-top centrifuge). Spin the tubes at 3,000 rpm for 30 minutes.

[0257] Return the unopened buckets to the cabinet. Remove the centrifuge tubes and pour the supernatant into a waste bottle for GTC lysis buffer.

[0258] Resuspend each pellet in 1 ml of Trizol (formulation of phenol and GTC cat no. 15596-026). The manufacturers guidelines recommend lysing cells by repetitive pipetting. Although this action alone will not lyse M. tuberculosis, it is important to completely resuspend the pellet in Trizol.

[0259] Transfer 1 ml of cells into a FastRNA tube and ribolyse it at power setting 6.5 for 45 seconds.

[0260] Leave the tube to incubate at room temperature for 5 minutes.

[0261] Remove the aqueous layer from the tube and add this to 200 μl of chloroform in a screw-capped eppendorf tube. Shake each tube vigorously for about 15 seconds. Incubate for 2-3 minutes at room temperature.

[0262] Spin the tube at 13,000 rpm for 15 minutes. Following centrifugation, the liquid separates into red phenol/chloroform phase, an interface, and a clear aqueous phase.

[0263] Carefully remove the aqueous phase and transfer it to a fresh eppendorf tube containing 500 μl of chloroform/isoamyl alcohol (24:1). Spin the tubes at 13,000 rpm for 15 minutes.

[0264] Transfer the aqueous phase to an eppendorf tube containing 50 μl of sodium acetate and 500 μl of isopropanol.

[0265] Surface decontaminate the eppendorf tube with 5% Hycolin for 5 minutes. Remove the tube from the CL3 laboratory and continue with the procedure in laboratory 157.

[0266] Steps performed at Containment level 2: Precipitate the RNA at −70° C. for at least 30 minutes-can do this step overnight.

[0267] Spin the precipitated RNA down at 13,000 rpm for 10 minutes. Remove the supernatant and wash the pellet in 70% ethanol. Repeat centrifugation.

[0268] Remove the 70% ethanol and air-dry the pellet. Dissolve the pellet in RNAse free water.

[0269] Freeze the RNA at −70° C. to store it.

EXAMPLE 3 Isolation of Genomic DNA From Mycobacterium tuberculosis Grown in Chemostat Culture. DNA Then Used to Generate Cy3 or Cy5 Labelled DNA for Use as a Control in Microarray Experiments

[0270] Materials and Methods

[0271] Beads 0.5 mm in diameter

[0272] Bead beater

[0273] Bench top centrifuge

[0274] Platform rocker

[0275] Heat block

[0276] Falcon 50 ml centrifuge tubes

[0277] Sorvall RC-5C centrifuge

[0278] 250 ml polypropylene centrifuge pots.

[0279] Screw capped eppendorf tubes

[0280] Pipettes 1 ml, 200 μl, 10 ml, 5 ml

[0281] Breaking buffer

[0282] 50 mM Tris HCl pH 8.0

[0283] 10 mM EDTA

[0284] 100 mM NaCl

[0285] Procedure

[0286] Mechanical Disruption of M. tuberculosis Cells

[0287] 150 ml of chemostat cells (O.D of 2.5 at 540 nm) are spun down at 15,000 rpm for minutes in 250 ml polypropylene pots using centrifuge Sorvall RC-5C.

[0288] The supernatant is discarded.

[0289] Cells are re-suspended in 5 ml of breaking buffer in a 50 ml Falcon tube and centrifuged at 15,000 rpm for a further 15 minutes.

[0290] The supernatant is removed and additional breaking buffer is added at a volume of 5 ml. Beads are used to disrupt the cells. These are used at a quantity of 1 ml of beads for 1 ml of cells. Place the sample into the appropriate sized chamber. Place in the bead beater and secure the outer unit (containing ice) and process at the desired speed for 30 seconds.

[0291] Allow the beads to settle for 10 minutes and transfer cell lysate to a 50 ml Falcon centrifuge tube

[0292] Wash beads with 2-5 ml of breaking buffer by pipetting washing buffer up and down over the beads.

[0293] Add this washing solution to the lysate in the falcon tube

[0294] Removal of Proteins and Cellular Components

[0295] Add 0.1 volumes of 10% SDS and 0.01 volumes proteinase K.

[0296] Mix by inversion and heat at 55° C. in a heat block for 2-3 hours

[0297] The resulting mix should be homogenous and viscous. Additional SDS may be added to assist here to bring the concentration up to 0.2%

[0298] Add an equal volume of phenol/chloroform/Isoamyl alcohol in the ratio: 25/24/1.

[0299] Gently mix on a platform rocker until homogenous

[0300] Spin down at 3,000 rpm for 20 minutes

[0301] Remove the aqueous phase and place in a fresh tube

[0302] Extract the aqueous phase with an equal volume of chloroform to remove traces of cell debris and phenol. Chloroform extractions may need to be repeated to remove all the debris.

[0303] Precipitate the DNA with 0.3 M sodium acetate and an equal volume of isopropanol.

[0304] Spool as much DNA as you can with a glass rod

[0305] Wash the spooled DNA in 70% ethanol followed by 100% ethanol

[0306] Leave to air dry

[0307] Dissolve the DNA in sterile deionised water (500 μl)

[0308] Allow DNA to dissolve at 4° C. for approximately 16 hours.

[0309] Add RNase 1 (500 U) to the dissolved DNA

[0310] Incubate for 1 hour at 37° C.

[0311] Re-extract with an equal volume of phenol/chloroform followed by a chloroform extraction and precipitate as before

[0312] Spin down the DNA at 13,000 rpm

[0313] Remove the supernatant and wash the pellet in 70% ethanol

[0314] Air dry

[0315] Dissolve in 200-500 μl of sterile water.

EXAMPLE 4 Preparation of Cy3 or Cy5 Labelled DNA from DNA

[0316] a) Prepare One Cy3 or one Cy5 Labelled DNA Sample per Microarray Slide

[0317] Each sample: DNA 2-5 μg Random primers (3 μg/μl) 1 μl H₂O to 41.5 μl

[0318] Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge.

[0319] Add to each: 10 × REact 2 buffer   5 μl dNTPs (5 mM dA/GTTP, 2 mM dCTP)   1 μl Cy3 OR Cy5 dCTP 1.5 μl Klenow (5 U/μl)   1 μl

[0320] Incubate at 37° C. in dark for 90 min.

[0321] b) Prehybridise Slide

[0322] Mix the prehybridisation solution in a Coplin jar and-incubate at 65° C. during the labelling reaction to equilibriate. Prehybridisation: 20 × SSC 8.75 ml (3.5 × SSC) 20% SDS 250 μl (0.1% SDS) BSA (100 mg/ml) 5 ml (10 mg/ml) H₂O to 50 ml

[0323] Incubate the microarray slide in the pre-heated prehybridisation solution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for 1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifuge slide in 50 ml centrifuge tube at 1,500 rpm for 5 min to dry. Store slide in dark, dust-free box until hybridisation (<1 h).

[0324] c) Purify Cy3/Cy5 Labelled DNA—Qiagen MinElute Purification

[0325] Combine Cy3 and Cy5 labelled DNA samples in single tube and add 500 μl Buffer PB.

[0326] Apply to MinElute column in collection tube and centrifuge at 13,000 rpm for 1 min.

[0327] Discard flow-through and place MinElute column back into same collection tube.

[0328] Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0329] Discard flow-through and place MinElute column back into same collection tube.

[0330] Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0331] Discard flow-through and place MinElute column back into same collection tube.

[0332] Centrifuge at 13,000 rpm for an additional 1 min to remove residual ethanol.

[0333] Place the MinElute column into a fresh 1.5 ml tube.

[0334] Add 10.5 μl H₂O to the centre of the membrane and allow to stand for 1 min.

[0335] Centrifuge at 13,000 rpm for 1 min.

EXAMPLE 5 Preparation of Cy3 or Cy5 Label cDNA from RNA

[0336] a) Prepare one Cy3 and One Cy5 Labelled cDNA Sample per Microarray Slide

[0337] Each sample: RNA 2-10 μg Random primers (3 μg/μl) 1 μl H₂O to 11 μl

[0338] Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge. Add to each: 5lFirst Strand Buffer   5 μl DTT (100 mM) 2.5 μl dNTPs (5 mM dA/G/TTP, 2 mM dCTP) 2.3 μl Cy3 OR Cy5 dCTP 1.7 μl SuperScript II (200 U/μl) 2.5 μl

[0339] Incubate at 25° C. in dark for 10 min followed by 42° C. in dark for 90 min.

[0340] b) Prehybridise Slide

[0341] Mix the prehybridisation solution in a Coplin jar and incubate at 65° C. during the labelling reaction to equilibrate.

[0342] Prehybridisation: 20 × SSC 8.75 ml (3.5 × SSC) 20% SDS 250 μl (0.1% SDS) BSA (100 mg/ml) 5 ml (10 mg/ml) H₂O to 50 ml

[0343] Incubate the microarray slide in the pre-heated prehybridisation solution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for 1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifuge slide in 50 ml centrifuge tube at 1500 rpm for 5 min to dry. Store slide in dark, dust-free box until hybridisation (<1 h).

[0344] c) Purify Cy3/Cy5 Labelled cDNA—Qiagen MinElute Purification

[0345] Combine Cy3 and Cy5 labelled DNA samples in single tube and add 250 μl Buffer PB.

[0346] Apply to MinElute column in collection tube and centrifuge at 13,000 rpm for 1 min.

[0347] Discard flow-through and place MinElute column back into same collection tube.

[0348] Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0349] Discard flow-through and place MinElute column back into same collection tube.

[0350] Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0351] Discard flow-through and place MinElute column back into same collection tube.

[0352] Centrifuge at 13,000 rpm for an additional 1 min to remove residual ethanol.

[0353] Place the MinElute column into a fresh 1.5 ml tube.

[0354] Add 10.5 μl H₂O to the centre of the membrane and allow to stand for 1 min.

[0355] Centrifuge at 13,000 rpm for 1 min.

EXAMPLE 6 Hybridise Slide with Cy3/Cy5 Labelled cDNA/DNA

[0356] Place the prehybridise microarray slide in the hybridisation cassette and add two 15 ml aliquots of H₂O to the wells in the cassette. Mix resuspended Cy3/Cy5 labelled cDNA/DNA sample with hybridisation solution. Hybridisation: Cy3/Cy5 labelled cDNA sample 10.5 ml 20 × SSC  3.2 ml (4 × SSC) 2% SDS  2.3 ml (0.3% SDS)

[0357] Heat hybridisation solution at 95° C. for 2 min. Do not snap cool on ice but allow to cool slightly and briefly centrifuge. Pipette the hybridisation solution onto the slide at the edge of the arrayed area avoiding bubble formation. Using forceps carefully drag the edge of a cover slip along the surface of the slide towards the arrayed area and into the hybridisation solution at the edge of the array. Carefully lower the cover slip down over the array avoiding any additional movement once in place. Seal the hybridisation cassette and submerge in a water bath at 60° C. for 16-20 h.

[0358] Wash slide.

[0359] Remove microarray slide from hybridisation cassette and initially wash slide carefully in staining trough of Wash A to remove cover slip. Once cover slip is displaced place slide(s) in slide rack and continue agitating in Wash A for a further 2 min. Wash A: 20 × SSC 20 ml (1 × SSC) 20% SDS 1 ml (0.05% SDS) H₂O to 400 ml

[0360] Transfer slide(s) to a clean slide rack and agitate in first trough of Wash B for 2 min. Wash in second trough of Wash B with agitation for 2 min. Wash B (x2): 20 × SSC 1.2 ml(0.06 × SSC) H₂O to 400 ml

[0361] Place slide into a 50 ml centrifuge tube and centrifuge at 1500 rpm for 5 mins to dry slide, and then scan fluorescence using a ScanArray 3000 dual-laser confocal scanner and analyse data.

EXAMPLE 7 Preparation of the Arrays

[0362] PCR-amplified products are generated from M. tuberculosis genomic DNA using ORF-specific primers. Each gene of the genome is represented. These are spotted in a grid onto a standard glass microscope slide using a BioRobotics microgrid robot (MWG Biotech) at a resolution of >4000 spots/cm².

EXAMPLE 8 Scanning and Analysis of Data

[0363] The slides were scanned using an Affymetrix 428 scanner.

[0364] Dual fluorescence is used, allowing simultaneous detection of two cDNA samples. The output of the arrays is read using a confocal laser scanner (Affymetrix 428 scanner from MWG Biotech). More detailed information can be found web site www.sghms.ac.uk/depts/medmicro/bugs; Mujumdar, R. B. (1993) Bioconjugate Chemistry, 4(2), pp. 105-111; Yu, H. (1994) Nucl. Acids Res. 22, pp.3226-3232; and Zhu, Z. (1994) Nucl. Acids Res. 22, pp. 3418-3422.

[0365] The raw data were initially analysed in software known as ImaGene, which was supplied with the scanner. The scanned images were then transferred to another software package known as GeneSpring. This is a very powerful tool, which draws information from many databases allowing the complete analysis of the expression of each gene.

EXAMPLE 9 Delete One or More of the Genes from M. tuberculosis in Order to Attenuate its Virulence While Retaining Immunogenicity

[0366] One or more genes that are identified may be disrupted using allelic exchange. In brief, the gene of interest is cloned with 1-2 kb of flanking DNA either side and is inactivated by deletion of part of the coding region and insertion of an antibiotic resistance marker, such as hygromycin.

[0367] The manipulated fragment is then transferred to a suitable suicide vector e.g. pPR23 and is transformed into the wild-type parent strain of M. tuberculosis. Mutants are recovered by selecting for antibiotic resistant strains. Genotypic analysis (Southern Blotting with a fragment specific to the gene of interest) is performed on the selected strains to confirm that the gene has been disrupted.

[0368] The mutant strain is then studied to determine the effect of the gene disruption on the phenotype. In order to use it as a vaccine candidate it would be necessary to demonstrated attenuated virulence. This can be done using either a guinea pig or mouse model of infection. Animals are infected with the mutant strain and the progression of disease is monitored by determining the bacterial load in different organs, in particular the lung and spleen, at specific time points post infection, typically up to 16 weeks.

[0369] Comparison is made to animals infected with the wild-type strain which should have a significantly higher bacterial load in the different organs. Long-term survival studies and histopathology can also be used to assess virulence and pathogenicity.

[0370] Once attenuated virulence has been established, protection and immunogenicity studies can be performed to assess the potential of the strain as a vaccine. Suitable references for allelic exchange and preparation of TB mutants are McKinney et al., 2000 and Pelicic et al., 1997, [1, 2].

EXAMPLE 10 Select One or More of the Genes Identifiable by the Present Invention, Which Encode Proteins that are Immunogenic, and Put Them into BCG or an Attenuated Strain of M. tuberculosis to Enhance its Overall Immunogenicity

[0371] The gene of interest is amplified from the M. tuberculosis genome by PCR. The amplified product is purified and cloned into a plasmid (pMV306) that integrates site specifically into the mycobacterial genome at the attachment site (attB) for mycobacteriophage L5 [3].

[0372] BCG is transformed with the plasmid by electroporation, which involves damaging the cell envelope with high voltage electrical pulses, resulting in uptake of the DNA. The plasmid integrates into the BCG chromosome at the attB site generating stable recombinants. Recombinants are selected and are checked by PCR or Southern blotting to ensure that the gene has been integrated. The recombinant strain is then used for protection studies.

EXAMPLE 11 Use of Recombinant Carriers such as Attenuated salmonella and the Vaccinia Virus to Express and Present TB Genes

[0373] One of the best examples of this type of approach is the use of Modified Vaccinia virus Ankara (MVA) [4]. The gene of interest is cloned into a vaccinia virus shuttle vector, e.g. pSC11. Baby Hamster Kidney (BHK) cells are then infected with wild-type MVA and are transfected with the recombinant shuttle vector. Recombinant virus is then selected using a suitable selection marker and viral plaques, selected and purified.

[0374] Recombinant virus is normally delivered as part of a prime-boost regime where animals are vaccinated initially with a DNA vaccine encoding the TB genes of interest under the control of a constitutive promoter. The immune response is boosted by administering recombinant MVA carrying the genes of interest to the animals at least 2 weeks later.

EXAMPLE 12 Sub-unit Vaccines Containing a Single Peptide/Protein or a Combination of Proteins

[0375] To prepare sub-unit vaccines with one or more peptides or proteins it is first of all necessary to obtain a supply of protein or peptide to prepare the vaccine. Up to now, this has mainly been achieved in mycobacterial studies by purifying proteins of interest from TB culture. However, it is becoming more common to clone the gene of interest and produce a recombinant protein.

[0376] The coding sequence for the gene of interest is amplified by PCR with restriction sites inserted at the N terminus and C terminus to permit cloning in-frame into a protein expression vector such as pET-15b. The gene is inserted behind an inducible promoter such as lacZ. The vector is then transformed into E. coli which is grown in culture. The recombinant protein is over-expressed and is purified.

[0377] One of the common purification methods is to produce a recombinant protein with an N-terminal His-tag. The protein can then be purified on the basis of the affinity of the His-tag for metal ions on a Ni-NTA column after which the His-tag is cleaved. The purified protein is then administered to animals in a suitable adjuvant [5].

EXAMPLE 13 Plasmid DNA Vaccines Carrying One or More of the Identified Genes

[0378] DNA encoding a specific gene is amplified by PCR, purified and inserted into specialised vectors developed for vaccine development, such as pVAX1. These vectors contain promoter sequences, which direct strong expression of the introduced DNA (encoding candidate antigens) in eukaryotic cells (eg. CMV or SV40 promoters), and polyadenlyation signals (eg. SV40 or bovine growth hormone) to stabilise the mRNA transcript.

[0379] The vector is transformed into E. coli and transformants are selected using a marker, such as kanamycin resistance, encoded by the plasmid. The plasmid is then recovered from transformed colonies and is sequenced to check that the gene of interest is present and encoded properly without PCR generated mutations. Large quantities of the plasmid is then produced in E. coli and the plasmid is recovered and purified using commercially available kits (e.g. Qiagen Endofree-plasmid preparation). The vaccine is then administered to animals for example by intramuscular injection in the presence or absence of an adjuvant.

EXAMPLE 14 Preparation of DNA Expression Vectors

[0380] DNA vaccines consist of a nucleic acid sequence of the present invention cloned into a bacterial plasmid. The plasmid vector pVAX1 is commonly used in the preparation of DNA vaccines. The vector is designed to facilitate high copy number replication in E. coli and high level transient expression of the peptide of interest in most mammalian cells (for details see manufacturers protocol for pVAX1 (catalog No. V260-20 www.invitrogen.com).

[0381] The vector contains the following elements:

[0382] Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a variety of mammalian cells

[0383] T7 promoter/priming site to allow in vitro transcription in the sense orientation and sequencing through the insert

[0384] Bovine growth hormone (BGH) polyadenylation signal for efficient transcription termination and polyadenylation of mRNA

[0385] Kanamycin resistance gene for selection in E. coli

[0386] A multiple cloning site

[0387] pUC origin for high-copy number replication and growth in E. coli

[0388] BGH reverse priming site to permit sequencing through the insert

[0389] Vectors may be prepared by means of standard recombinant techniques which are known in the art, for example Sambrook et al. (1989). Key stages in preparing the vaccine are as follows:

[0390] The gene of interest is ligated into pVAX1 via one of the multiple cloning sites

[0391] The ligation mixture is then transformed into a competent E. coli strain (e.g. TOP1 0) and LB plates containing 50 μg/ml kanamycin are used to select transformants.

[0392] Clones are selected and may be sequenced to confirm the presence and orientation of the gene of interest.

[0393] Once the presence of the gene has been verified, the vector can be used to transfect a mammalian cell line to check for protein expression. Methods for transfection are known in the art and include, for example, electroporation, calcium phosphate, and lipofection.

[0394] Once peptide expression has been confirmed, large quantities of the vector can be produced and purified from the appropriate cell host, e.g E. coli.

[0395] pVAX1 does not integrate into the host chromosome. All non-essential sequences have been removed to minimise the possibility of integration. When constructing a specific vector, a leader sequence may be included to direct secretion of the encoded protein when expressed inside the eukaryotic cell.

[0396] Other examples of vectors that have been used are V1Jns.tPA and pCMV4 (Lefevre et al., 2000 and Vordermeier et al., 2000).

[0397] Expression vectors may be used that integrate into the genome of the host, however, it is more common and more preferable to use a vector that does not integrate. The example provided, pVAX1, does not integrate. Integration would lead to the generation of a genetically modified host which raises other issues.

EXAMPLE 15 RNA Vaccine

[0398] As discussed on page 15 of U.S. Pat. No. 5,783,386, one approach is to introduce RNA directly into the host.

[0399] Thus, the vector construct (Example 14) may be used to generate RNA in vitro and the purified RNA then injected into the host. The RNA would then serve as a template for translation in the host cell. In this embodiment, integration would not normally occur.

[0400] Another option is to use an infectious agent such as the retroviral genome carrying RNA corresponding to the gene of interest. In this embodiment, integration into the host genome will occur.

[0401] Another option is the use of RNA replicon vaccines which can be derived from virus vectors such as Sindbis virus or Semliki Forest virus. These vaccines are self-replicating and self-limiting and may be administered as either RNA or DNA which is then transcribed into RNA replicons in vivo. The vector eventually causes lysis of the transfected cells thereby reducing concerns about integration into the host genome. Protocols for RNA vaccine construction are detailed in Cheng et al., (2001).

EXAMPLE 16 Diagnostic Assays Based on Assessing T Cell Responses

[0402] For a diagnostic assay based on assessing T cell responses it would be sufficient to obtain a sample of blood from the patient. Mononuclear cells (monocytes, T and B lymphocytes) can be separated from the blood using density gradients such as Ficoll gradients.

[0403] Both monocytes and B-lymphocytes are both able to present antigen, although less efficiently than professional antigen presenting cells (APCs) such as dendritic cells. The latter are more localised in lymphoid tissue.

[0404] The simplest approach would be to add antigen to the separated mononuclear cells and incubate for a week and then assess the amount of proliferation. If the individual had been exposed to the antigen previously through infection, then T-cell closes specific to the antigen should be more prevalent in the sample and should respond.

[0405] It is also possible to separate the different cellular populations should it be desired to control the ratio of T cells to APC's.

[0406] Another variation of this type of assay is to measure cytokine production by the responding lymphocytes as a measure of response. The ELISPOT assay described below in Example 17 is a suitable example of this variation.

EXAMPLE 17 Detection of Latent Mycobacteria

[0407] A major problem for the control of tuberculosis is the presence of a large reservoir of asymptomatic individuals infected with tubercle bacilli. Dormant bacilli are more resistant to front-line drugs.

[0408] The presence of latent mycobacteria-associated antigen may be detected indirectly either by detecting antigen specific antibody or T-cells in blood samples.

[0409] The following method is based on the method described in Lalvani et al. (2001) in which a secreted antigen, ESAT-6, was identified as being expressed by members of the M. tuberculosis complex but is absent from M. Bovis BCG vaccine strains and most environmental mycobacteria. 60-80% of patients also have a strong cellular immune response to ESAT-6. An ex-vivo ELISPOT assay was used to detect ESAT-6 specific T cells.

[0410] As applied to the present invention:

[0411] A 96 well plate is coated with cytokine (e.g. interferon-y, IL-2) -specific antibody. Peripheral blood monocytes are then isolated from patient whole blood and are applied to the wells.

[0412] Antigen (ie. one of the peptides, fragments, derivatives or variants of the present invention) is added to stimulate specific T cells that may be present and the plates are incubated for 24 h. The antigen stimulates cytokine production which then binds to the specific antibody.

[0413] The plates are washed leaving a footprint where antigen-specific T cells were present.

[0414] A second antibody coupled with a suitable detection system, e.g. enzyme, is then added and the number of spots are enumerated after the appropriate substrate has been added.

[0415] The number of spots, each corresponding to a single antigen-specific T cell, is related to the total number of cells originally added.

[0416] The above Example also describes use of an antigen that may be used to distinguish TB infected individuals from BCG vaccinated individuals. This could be used in a more discriminative diagnostic assay.

EXAMPLE 18 In vitro Model for Mycobacterial Persistence Under the Joint Conditions of Carbon-Starvation and Oxygen-Limitation (a Variation on Examples 1-8)

[0417] Materials and Methods

[0418] Studies were performed with M. tuberculosis strain H37Rv (NCTC cat. No. 7416)—a representative strain of M. tuberculosis. Stock cultures were grown on Middlebrook 7H10+OADC for 3 weeks at 37±2° C.

[0419] Culture Medium

[0420] Persistence cultures were established in Middlebrook 7H9 medium supplemented with Middlebrook ADC enrichment, 0.2% Tween 80 and 0.2% glycerol (see below). The medium was prepared with high quality water from a Millipore water purification system and filter sterilised by passage through a 0.1 μm pore size cellulose acetate membrane filter capsule (Sartorius Ltd). The pH was adjusted to 6.6 with concentrated hydrochloric acid.

[0421] Middlebrook 7H10+OADC agar was used to prepare inoculum cultures, enumerate the number of culturable bacteria in samples, and to assess culture purity.

[0422] Culture System

[0423] We used the culture system described in WO00/52139, operated as a batch fermenter, for this Example.

[0424] Culture experiments were performed in a one litre glass vessel operated at a working volume of 750 ml. The culture was agitated by a magnetic bar placed in the culture vessel coupled to a magnetic stirrer positioned beneath the vessel. Culture conditions were continuously monitored by an Anglicon Microlab Fermentation System (Brighton Systems, Newhaven), linked to sensor probes inserted into the culture through sealed ports in the top plate. The oxygen concentration was monitored with a galvanic oxygen electrode (Uniprobe, Cardiff) and was controlled through sparging the culture with a mixture of air and oxygen free-nitrogen. Temperature was monitored by an Anglicon temperature probe, and maintained by a heating pad positioned beneath the culture vessel. Culture pH was measured using an Ingold pH electrode (Mettler-Toledo, Leicester).

[0425] Inoculation and Culture

[0426] The vessel was filled with 750 ml of sterile culture medium and parameters were allowed to stabilise at 37° C.±2° C., pH 6.9±0.3 and a dissolved oxygen tension of approximately 70% air saturation. A dense inoculum suspension was prepared by resuspending Middlebrook agar cultures, grown at 37±2° C. for 3 week, in sterile deionised water. The inoculum was aseptically transferred to the culture vessel, to provide an initial culture turbidity of approximately 0.25 at 540 nm. The culture was maintained at 37° C. with an agitation rate of 500 to 750 rpm. After inoculation, the dissolved oxygen tension (DOT) of the culture was maintained at approximately 40% air saturation at 37° C. until the culture had entered early exponential growth. The DOT was then lowered in increments down to 1% air saturation over a six day period (FIG. 3). The culture was then maintained at a DOT of 0-5% until 50 days after inoculation and samples were removed regularly to monitor growth and survival, nutrient utilisation and gene expression.

[0427] Growth and Survival

[0428] Bacterial growth and survival was assessed by determining the number of viable cells in the culture system at specific time points. This was achieved by preparing a decimal dilution series of the sample in sterile water and plating 100(I aliquots onto Middlebrook 7H10+OADC plates. The plates were incubated at 37° C. for up to 4 weeks before enumerating the number of colonies formed.

[0429] Nutrient Utilisation

[0430] Glycerol is the primary carbon and energy source present in Middlebrook 7H9 medium with ADC, 0.2% Tween and Glycerol. The rate at which glycerol was utilised was determined using the Glycerol Determination Kit Cat. No. 148 270 Boehringer Mannheim.

[0431] Microarray Experiments

[0432] RNA was extracted from culture samples collected at different time points during the experiment. A fluorescently-labelled cDNA was then transcribed from each sample of RNA. The cDNA was labelled by the incorporation of either Cy3 or Cy5 labelled dCTP (Dyes are supplied by Amersham Pharmacia Biotech).

[0433] Whole M. tuberculosis genome arrays were prepared from M. tuberculosis genomic DNA using ORF-specific primers. PCR products corresponding to each ORF were spotted in a grid onto a standard glass microscope slide using a BioRobotics microgrid robot (MWG Biotech) at a resolution of >4000 spots/cm². Arrays were supplied by Dr P Butcher, St George's Hospital Medical School London.

[0434] In each microarray experiment a whole genome array was hybridised with labelled cDNA from one culture sample (Test sample). Each array was also hybridised with control DNA incorporating a different Cy dye and prepared from DNA extracted from M. tuberculosis strain H37Rv (control sample). Each array was scanned, using an Affymetrix 428 scanner, at two different wavelengths corresponding to the excitation maxima of each dye and the intensity of the emitted light was recorded. The raw data was processed by ImaGene software before performing comparative analysis using GeneSpring.

[0435] Results

[0436] Analysis of viable count data indicated that the culture grew exponentially until 10 to 12 days post infection (FIG. 4). As the culture entered stationary phase, viability started to decline and continued to decline steadily over the duration of the study. After 40 days in stationary phase, approximately 0.1% of the culture was still culturable on Middlebrook agar. The rate of glycerol utilisation was slower than observed in the culture established under aerobic conditions, indicating that the metabolic activity of the low-oxygen culture was restricted by limited oxygen availability. Nevertheless, the principal carbon and energy source was depleted within 15 days after inoculation (FIG. 2).

[0437] Samples were collected for microarray analysis as outlined. The gene expression profiles for samples collected at day 5 and 50 were compared. Three arrays were prepared for each sample and the test data was normalised against the control data on each chip. The normalised data for each set of arrays was then averaged and the two data sets were compared. Those genes that were expressed 5-fold higher at day 5 relative to day 50 were selected. The gene list was compared with the list generated from the carbon starvation model (ie. Table 2), and a sub-list of genes which were only down-regulated under the combined conditions of carbon starvation and oxygen-limitation was generated (see Table 3).

[0438] Liquid medium formulation for persistence cultures—Middlebrook 7H9 medium supplemented with ADC, 0.2% Tween 80 and 0.2% Glycerol Composition per liter Na₂HPO₄ 2.5 g KH₂PO₄ 1.0 g Monosodium glutamate 0.5 g (NH4)₂SO₄ 0.5 g Sodium citrate 0.1 g MgSO₄.7H₂O 0.05 g Ferric ammonium citrate 0.04 g CuSO₄.5H₂O 1.0 mg Pyridoxine 1.0 mg ZnSO₄.7H₂O 1.0 mg Biotin 0.5 mg CaCl₂.2H₂0 0.5 mg Middlebrook ADC enrichment 100 ml Glycerol 2.0 ml Tween 80 2.0 ml Middlebrook ADC enrichment - per 100 ml Bovine serum albumin 5.0 g Glucose 2.0 g Catalase 3.0 mg

[0439] Microarray Protocols

[0440] 1. RNA Extraction from M. tuberculosis for Microarray Analysis

[0441] Materials and Methods

[0442] Trizol (Life Technologies)—formulation of phenol and guanidine thiocyanate.

[0443] GTC lysis solution containing: 5 M guanidine thiocyanate, 0.5% N-lauryl sarcosine, 25 mM tri-sodium citrate, 0.1 M 2-mercaptoethanol, and 0.5% Tween 80.

[0444] Chloroform

[0445] Isopropanol

[0446] 3 M sodium acetate

[0447] *70% Ethanol

[0448] microfuge

[0449] ribolyser

[0450] Sterile plasticware-Falcon tubes, screw capped eppendorfs, gilson tips—all RNase free

[0451] Glassware—baked at 160° C. for at least 16 hours

[0452] Method

[0453] Steps performed at Containment level 3; within a Class III microbiological safety cabinet.

[0454] Remove 10 or 20 ml of culture (10⁹/ml) and immediately add this to 4 volumes of GTC lysis buffer in a plastic specimen pot. Seal the pot tightly.

[0455] Incubate the cells in GTC lysis buffer for 1 hour at room temperature. Surface decontaminate the plastic pot with 5% Hycolin for 5 minutes. Transfer the sample to the pass box and place it into a plastic carry tin with a sealable lid. Close the container securely and transport it to a non-toxic cabinet CL3 cabinet.

[0456] Equally distribute the lysis mixture between Falcon tubes. Place these tubes into centrifuge buckets and seal the buckets tightly. Surface-decontaminate the buckets for 5 minutes with 5% Hycolin. Then transferthem to the centrifuge (Baird and Tatlock Mark IV refrigerated bench-top centrifuge). Spin the tubes at 3,000 rpm for 30 minutes.

[0457] Return the unopened buckets to the cabinet. Remove the centrifuge tubes and pour the supernatant into a waste bottle for GTC lysis buffer.

[0458] Resuspend each pellet in 5 ml of Trizol (formulation of phenol and GTC cat No. 15596-026). The manufacturers guidelines recommend lysing cells by repetitive pipetting. Although this action alone will not lyse M. tuberculosis, it is important to completely resuspend the pellet in Trizol.

[0459] Transfer 1 ml of cells into each FastRNA tube and ribolyse them at power setting 6.5 for 45 seconds.

[0460] Leave the tubes to incubate at room temperature for 5 minutes.

[0461] Remove the aqueous layer from each tube and add this to 200 μl of chloroform in a screw-capped eppendorf tube. Shake each tube vigorously for about 15 seconds. Incubate for 2-3 minutes at room temperature.

[0462] Spin the tubes at 13,000 rpm for 15 minutes. Following centrifugation, the liquid separates into red phenol/chloroform phase, an interface, and a clear aqueous phase.

[0463] Carefully remove the aqueous phase and transfer it to fresh eppendorf tubes containing 500 μl of chloroform/isoamyl alcohol (24:1). Spin the tubes at 13,000 rpm for 15 minutes.

[0464] Transfer the aqueous phase to eppendorf tubes containing 50 μl of sodium acetate and 500 μl of isopropanol.

[0465] Surface decontaminate the eppendorf tubes with 5% Hycolin for 5 minutes. Remove the tubes from the CL3 laboratory and continue with the procedure in laboratory 157.

[0466] Steps performed at Containment level 2:

[0467] Precipitate the RNA at −70° C. for at least 30 minutes (optionally overnight).

[0468] Spin the precipitated RNA down at 13,000 rpm for 10 minutes. Remove the supernatant and wash the pellet in 70% ethanol. Repeat centrifugation.

[0469] Remove the 70% ethanol and air-dry the pellet. Dissolve the pellet in RNAse free water.

[0470] Freeze the RNA at −70° C. to store it.

[0471] 2. Isolation of Genomic DNA from Mycobacterium tuberculosis Grown in Chemostat Culture. DNA Then Used to Generate Cy3 or Cy5 Labelled DNA for Use as a Control in Microarray Experiments

[0472] Materials and Methods

[0473] Beads 0.5 mm in diameter

[0474] Bead beater

[0475] Bench top centrifuge

[0476] Platform rocker

[0477] Heat block

[0478] Falcon 50 ml centrifuge tubes

[0479] Sorvall RC-5C centrifuge

[0480] 250 ml polypropylene centrifuge pots.

[0481] Screw capped eppendorf tubes

[0482] Pipettes 1 ml, 200 μl, 10 ml, 5 ml

[0483] Breaking buffer

[0484] 50 mM Tris HCL pH 8.0

[0485] 10 mM EDTA

[0486] 100 mM NaCl

[0487] Procedure

[0488] Mechanical Disruption of Mtb Cells

[0489] 150 ml of chemostat cells (O.D of 2.5 at 540 nm) are spun down at 15,000 rpm for 15 minutes in 250 ml polypropylene pots using centrifuge Sorvall RC-5C.

[0490] The supernatant is discarded.

[0491] Cells are re-suspended in 5 ml of breaking buffer in a 50 ml Falcon tube and centrifuged at 15,000 rpm for a further 15 minutes.

[0492] The supernatant is removed and additional breaking buffer is added at a volume of 5 ml. Beads are used to disrupt the cells. These are used at a quantity of 1 ml of beads for 1 ml of cells. Place the sample into the appropriate sized chamber. Place in the bead beater and secure the outer unit (containing ice) and process at the desired speed for 30 seconds.

[0493] Allow the beads to settle for 10 minutes and transfer cell lysate to a 50 ml Falcon centrifuge tube

[0494] Wash beads with 2-5 ml of breaking buffer by pipetting washing buffer up and down over the beads.

[0495] Add this washing solution to the lysate in the falcon tube

[0496] Removal of Proteins and Cellular Components.

[0497] Add 0.1 volumes of 10% SDS and 0.01 volumes proteinase K.

[0498] Mix by inversion and heat at 55° C. in a heat block for 2-3 hours

[0499] The resulting mix should be homogenous and viscous. If it isn't then add more SDS to bring the concentration up to 0.2%

[0500] Add an equal volume of phenol/chloroform/Isoamyl alcohol in the ratio: 25/24/1.

[0501] Gently mix on a platform rocker until homogenous

[0502] Spin down at 3,000 rpm for 20 minutes

[0503] Remove the aqueous phase and place in a fresh tube

[0504] Extract the aqueous phase with an equal volume of chloroform to remove traces of cell debris and phenol. Chloroform extractions may need to be repeated to remove all the debris.

[0505] Precipitate the DNA with 0.3 M sodium acetate and an equal volume of isopropanol.

[0506] Spool as much DNA as you can with a glass rod

[0507] Wash the spooled DNA in 70% ethanol followed by 100% ethanol

[0508] Leave to air dry

[0509] Dissolve the DNA in sterile deionised water (500 μl)

[0510] Allow DNA to dissolve at 4° C. for approximately 16 hours.

[0511] Add RNase 1 (500 U) to the dissolved DNA

[0512] Incubate for 1 hour at 37° C.

[0513] Re-extract with an equal volume of phenol/chloroform followed by a chloroform extraction and precipitate as before

[0514] Spin down the DNA at 13,000 rpm

[0515] Remove the supernatant and wash the pellet in 70% ethanol

[0516] Air dry

[0517] Dissolve in 200-500 μl of sterile water.

[0518] 3. Preparation of Cy3 or Cy5 Labelled DNA from DNA

[0519] a) Prepare One Cy3 or One Cy5 Labelled DNA Sample per Microarray Slide. Each sample: DNA 2-5 μg Random primers (3 μg/μl) 1 μl H₂O to 41.5 μl

[0520] Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge. Add to each: 10 * REact 2 buffer   5 μl dNTPs (5 mM dA/G/TTP, 2 mM dCTP)   1 μl Cy3 OR Cy5 dCTP 1.5 μl Klenow (5 U/μl)   1 μl

[0521] Incubate at 37° C. in dark for 90 min.

[0522] b) Prehybridise Slide

[0523] Mix the prehybridisation solution in a Coplin jar and incubate at 65° C. during the labelling reaction to equilibriate. Prehybridisation: 20 * SSC 8.75 ml(3.5 * SSC) 20% SDS 250 μl(0.1% SDS) BSA (100 mg/ml) 5 ml(10 mg/ml) H₂O to 50 ml

[0524] Incubate the microarray slide in the pre-heated prehybridisation solution at 65° C. for min. Rinse slide thoroughly in 400 ml H₂O for 1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifuge slide in 50 ml centrifuge tube at 1,500 rpm for 5 min to dry. Store slide in dark, dust-free box until hybridisation (<1 h).

[0525] c) Purify Cy3/Cy5 Labelled DNA—Qiagen MinElute Purification

[0526] Combine Cy3 and Cy5 labelled DNA samples in single tube and add 500 μl Buffer PB.

[0527] Apply to MinElute column in collection tube and centrifuge at 13,000 rpm for 1 min.

[0528] Discard flow-through and place MinElute column back into same collection tube.

[0529] Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0530] Discard flow-through and place MinElute column back into same collection tube.

[0531] Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0532] Discard flow-through and place MinElute column back into same collection tube.

[0533] Centrifuge at 13,000 rpm for an additional 1 min to remove residual ethanol.

[0534] Place the MinElute column into a fresh 1.5 ml tube.

[0535] Add 10.5 μl H₂O to the centre of the membrane and allow to stand for 1 min.

[0536] Centrifuge at 13,000 rpm for 1 min.

[0537] 4. Preparation of Cy3 or Cy5 Label cDNA from RNA.

[0538] a) Prepare One Cy3 and One Cy5 Labelled cDNA Sample per Microarray Slide. Each sample: RNA 2-10 μg Random primers (3 μg/μl) 1 μl H₂O to 11 μl

[0539] Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge. Add to each: 5 * First Strand Buffer   5 μl DTT (100 mM) 2.5 μl dNTPs (5 mM dA/G/TTP, 2 mM dCTP) 2.3 μl Cy3 OR Cy5 dCTP 1.7 μl SuperScript II (200 U/μl) 2.5 μl

[0540] Incubate at 25° C. in dark for 10 min followed by 42° C. in dark for 90 min.

[0541] b) Prehybridise Slide

[0542] Mix the prehybridisation solution in a Coplin jar and incubate at 65° C. during the labelling reaction to equilibrate.

[0543] Prehybridisation: 20 * SSC 8.75 ml (3.5 * SSC) 20% SDS 250 μl (0.1% SDS) BSA (100 mg/ml) 5 ml (10 mg/ml) H₂O to 50 ml

[0544] Incubate the microarray slide in the pre-heated prehybridisation solution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for 1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifuge slide in 50 ml centrifuge tube at 1500 rpm for 5 min to dry. Store slide in dark, dust-free box until hybridisation (<l h).

[0545] c) Purify Cy3/Cy5 Labelled cDNA—Qiagen MinElute Purification

[0546] Combine Cy3 and Cy5 labelled DNA samples in single tube and add 250 μl Buffer PB.

[0547] Apply to MinElute column in collection tube and centrifuge at 13,000 rpm for 1 min.

[0548] Discard flow-through and place MinElute column back into same collection tube.

[0549] Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0550] Discard flow-through and place MinElute column back into same collection tube.

[0551] Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000 rpm for 1 min.

[0552] Discard flow-through and place MinElute column back into same collection tube.

[0553] Centrifuge at 13,000 rpm for an additional 1 min to remove residual ethanol.

[0554] Place the MinElute column into a fresh 1.5 ml tube.

[0555] Add 10.5 μl H₂O to the centre of the membrane and allow to stand for 1 min.

[0556] Centrifuge at 13,000 rpm for 1 min.

[0557] 5. Hybridise Slide with Cy3/Cy5 Labelled cDNA/DNA

[0558] Place the prehybridise microarray slide in the hybridisation cassette and add two 15 μl aliquots of H₂O to the wells in the cassette. Mix resuspended Cy3/Cy5 labelled cDNA sample with hybridisation solution. Hybridisation: Cy3/Cy5 labelled cDNA sample 10.5 μl 20 × SSC  3.2 μl (4 × SSC) 2% SDS  2.3 μl (0.3% SDS)

[0559] Heat hybridisation solution at 95° C. for 2 min. Do NOT snap cool on ice but allow to cool slightly and briefly centrifuge. Pipette the hybridisation solution onto the slide at the edge of the arrayed area avoiding bubble formation. Using forceps carefully drag the edge of a cover slip along the surface of the slide towards the arrayed area and into the hybridisation solution at the edge of the array. Carefully lower the cover slip down over the array avoiding any additional movement once in place. Seal the hybridisation cassette and submerge in a water bath at 65° C. for 16-20 hours.

[0560] Wash Slide

[0561] Remove microarray slide from hybridisation cassette and initially wash slide carefully in staining trough of Wash A preheated to 65° C. to remove cover slip. Once cover slip is displaced place slide(s) in slide rack and continue agitating in Wash A for a further 2 min. Wash A: 20 × SSC 20 ml (1 × SSC) 20% SDS  1 ml (0.05% SDS) H₂O to 400 ml

[0562] Transfer slide(s) to a clean slide rack and agitate in first trough of Wash B for 2 min. Wash in second trough of Wash B with agitation for 2 min. Wash B (x2): 20 × SSC 1.2 ml (0.06 × SSC) H₂O to 400 ml

[0563] Place slide into a 50 ml centrifuge tube and centrifuge at 1500 rpm for 5 mins to dry slide and then scan fluorescence.

[0564] The genes identified in accordance with Example 18 are listed in Table 3. Those genes overlapping with Table 2 have been omitted from Table 3. The coding sequences for these genes (nucleic acid sequences are given from the transcription start site to the stop codon) and corresponding amino acid sequences are listed in the accompanying Sequence Listing starting from SEQ ID NO. 21 et seq (amino acid sequences are followed immediately by corresponding coding sequences for each gene). TABLE 3 Genes down-regulated during survival under the combined conditions of carbon starvation and oxygen limitation (excluding those genes which are down-regulated in response to the single stimulus of carbon starvation - Table 2) Genes Fold- Assigned function SEQ ID NO: Rv0511; 5.7679 Uroporphyrin-III C-methyltransferase HEMD (uroporphyrinogen III 21;22 cysG (9015) methylase) (urogen III methylase) (SUMT) (urogen III methylase) Rv1079; 6.1459 Cystathionine gamma-synthase METB (CGS) (O-succinylhomoserine 23;24 metB (5G11) [Thiol]-lyase)23;24 Rv0206c; 5.3012 Transmembrane transport protein MMPL3 25;26 mmpL3 (1K8) Rv2942; 5.0236 Transmembrane transport protein MMPL7 27;28 mmpL7 (2K8) Rv0055; 5.0654 30S ribosomal protein S18-1 RPSR1 29;30 rpsR (1G7) Rv0144; 8.3578 Transcriptional regulatory protein 31;32 (8P3) Rv0198c; 5.4541 Zinc metalloprotease 33;34 (1K7) Rv0361; 5.8502 Membrane protein 35;36 (2L23) Rv0635; 7.212 37;38 (4L23) Rv0805; 6.3055 39;40 (3023) Rv1871c; 5.495 41;42 (10O15) Rv3599c; 19.6683 43;44 (11C1) Rv3661; (2C23) 5.2733 45;46

[0565]

1 46 1 380 PRT Mycobacterium tuberculosis 1 Met Thr Arg Arg Thr Gly Gln Arg Trp Arg Gly Thr Leu Pro Gly Arg 1 5 10 15 Arg Pro Trp Thr Arg Pro Ala Pro Ala Thr Cys Arg Arg His Leu Ala 20 25 30 Phe Val Glu Leu Arg His Tyr Phe Ala Arg Val Met Ser Ser Ala Ile 35 40 45 Gly Ser Val Ala Arg Trp Ile Val Pro Leu Leu Gly Val Ala Ala Val 50 55 60 Ala Ser Ile Gly Val Ile Ala Asp Pro Val Arg Val Val Arg Ala Pro 65 70 75 80 Ala Leu Ile Leu Val Asp Ala Ala Asn Pro Leu Ala Gly Lys Pro Phe 85 90 95 Tyr Val Asp Pro Ala Ser Ala Ala Met Val Ala Ala Arg Asn Ala Asn 100 105 110 Pro Pro Asn Ala Glu Leu Thr Ser Val Ala Asn Thr Pro Gln Ser Tyr 115 120 125 Trp Leu Asp Gln Ala Phe Pro Pro Ala Thr Val Gly Gly Thr Val Ala 130 135 140 Arg Tyr Thr Gly Ala Ala Gln Ala Ala Gly Ala Met Pro Val Leu Thr 145 150 155 160 Leu Tyr Gly Ile Pro His Arg Asp Cys Gly Ser Tyr Ala Ser Gly Gly 165 170 175 Phe Ala Thr Gly Thr Asp Tyr Arg Gly Trp Ile Asp Ala Val Ala Ser 180 185 190 Gly Leu Gly Ser Ser Pro Ala Thr Ile Ile Val Glu Pro Asp Ala Leu 195 200 205 Ala Met Ala Asp Cys Leu Ser Pro Asp Gln Arg Gln Glu Arg Phe Asp 210 215 220 Leu Val Arg Tyr Ala Val Asp Thr Leu Thr Arg Asp Pro Ala Ala Ala 225 230 235 240 Val Tyr Val Asp Ala Gly His Ser Arg Trp Leu Ser Ala Glu Ala Met 245 250 255 Ala Ala Arg Leu Asn Asp Val Gly Val Gly Arg Ala Arg Gly Phe Ser 260 265 270 Leu Asn Val Ser Asn Phe Tyr Thr Thr Asp Glu Glu Ile Gly Tyr Gly 275 280 285 Glu Ala Ile Ser Gly Leu Thr Asn Gly Ser His Tyr Val Ile Asp Thr 290 295 300 Ser Arg Asn Gly Ala Gly Pro Ala Pro Asp Ala Pro Leu Asn Trp Cys 305 310 315 320 Asn Pro Ser Gly Arg Ala Leu Gly Ala Pro Pro Thr Thr Ala Thr Ala 325 330 335 Gly Ala His Ala Asp Ala Tyr Leu Trp Ile Lys Arg Pro Gly Glu Ser 340 345 350 Asp Gly Thr Cys Gly Arg Gly Glu Pro Gln Ala Gly Arg Phe Val Ser 355 360 365 Gln Tyr Ala Ile Asp Leu Ala His Asn Ala Gly Gln 370 375 380 2 1140 DNA Mycobacterium tuberculosis 2 atgacgcgtc ggactgggca gcgatggcgc gggactctgc ccgggcgccg gccttggaca 60 cggccagcgc ccgccacctg tcgtcggcat ttggcgtttg tcgaattgcg gcattatttt 120 gctcgggtga tgtcatcagc tattggttcg gtcgcgcggt ggatagtccc cctcctgggg 180 gttgcagccg ttgcttccat cggtgttatc gcggacccgg tgcgggtcgt tcgggccccg 240 gcgttgatcc tggtcgatgc ggcaaacccg ctggccggaa agcccttcta cgtcgatccc 300 gcctcggcgg ccatggtcgc cgcgcgcaac gccaacccgc cgaacgccga gctgacctcc 360 gtcgccaaca ccccgcagtc ctactggctc gaccaggcat tcccgccggc gaccgtcggc 420 ggcacggttg ccaggtacac cggagcggcg caggcggccg gcgccatgcc ggttctgacg 480 ctgtatggaa tcccccatcg cgactgcggt agctacgcat ccggtgggtt cgcgacgggc 540 actgattacc gcgggtggat cgacgctgtc gcatccggcc tgggctcatc gccggcgacg 600 atcatcgtcg aacccgatgc gctggccatg gccgactgcc tgtcgcctga ccagcgccag 660 gaacgtttcg acttggtgcg ctacgccgtc gacacgctga cccgcgaccc ggccgctgcc 720 gtgtacgtcg atgcggggca ttcgcgctgg ctgagcgccg aggcaatggc cgccaggctc 780 aacgatgtcg gtgtgggccg cgcgcgcggg tttagcctca acgtctcgaa cttctacacc 840 accgatgagg aaatcggcta tggcgaggcg atttcggggc tcacgaacgg ttcgcattac 900 gtgatcgaca cgtcgcgcaa cggcgccgga cccgcgcccg acgccccgct caactggtgt 960 aaccccagcg gccgcgccct gggcgcaccg cccaccacgg cgaccgcggg cgcgcacgcc 1020 gacgcttacc tgtggatcaa acgtcccggg gaatcggacg gaacctgcgg tcgcggggag 1080 cctcaggcgg gtcggttcgt tagccagtac gccatcgatc tggcccacaa cgccggccag 1140 3 417 PRT Mycobacterium tuberculosis 3 Val Asn Leu Gln Leu Phe Leu Leu Leu Ile Val Val Val Thr Ala Leu 1 5 10 15 Ala Phe Asp Phe Thr Asn Gly Phe His Asp Thr Gly Asn Ala Met Ala 20 25 30 Thr Ser Ile Ala Ser Gly Ala Leu Ala Pro Arg Val Ala Val Ala Leu 35 40 45 Pro Ala Val Leu Asn Leu Ile Gly Ala Phe Leu Ser Thr Ala Val Ala 50 55 60 Ala Thr Ile Ala Lys Gly Leu Ile Asp Ala Asn Leu Val Thr Leu Glu 65 70 75 80 Leu Val Phe Ala Gly Leu Val Gly Gly Ile Val Trp Asn Leu Leu Thr 85 90 95 Trp Leu Leu Gly Ile Pro Ser Ser Ser Ser His Ala Leu Ile Gly Gly 100 105 110 Ile Val Gly Ala Thr Ile Ala Ala Val Gly Leu Arg Gly Val Ile Trp 115 120 125 Ser Gly Val Val Ser Lys Val Ile Val Pro Ala Val Val Ala Ala Leu 130 135 140 Leu Ala Thr Leu Val Gly Ala Val Gly Thr Trp Leu Val Tyr Arg Thr 145 150 155 160 Thr Arg Gly Val Ala Glu Lys Arg Thr Glu Arg Gly Phe Arg Arg Gly 165 170 175 Gln Ile Gly Ser Ala Ser Leu Val Ser Leu Ala His Gly Thr Asn Asp 180 185 190 Ala Gln Lys Thr Met Gly Val Ile Phe Leu Ala Leu Met Ser Tyr Gly 195 200 205 Ala Val Ser Thr Thr Ala Ser Val Pro Pro Leu Trp Val Ile Val Ser 210 215 220 Cys Ala Val Ala Met Ala Ala Gly Thr Tyr Leu Gly Gly Trp Arg Ile 225 230 235 240 Ile Arg Thr Leu Gly Lys Gly Leu Val Glu Ile Lys Pro Pro Gln Gly 245 250 255 Met Ala Ala Glu Ser Ser Ser Ala Ala Val Ile Leu Leu Ser Ala His 260 265 270 Phe Gly Tyr Ala Leu Ser Thr Thr Gln Val Ala Thr Gly Ser Val Leu 275 280 285 Gly Ser Gly Val Gly Lys Pro Gly Ala Glu Val Arg Trp Gly Val Ala 290 295 300 Gly Arg Met Val Val Ala Trp Leu Val Thr Leu Pro Leu Ala Gly Leu 305 310 315 320 Val Gly Ala Phe Thr Tyr Gly Leu Val His Phe Ile Gly Gly Tyr Pro 325 330 335 Gly Ala Ile Leu Gly Phe Ala Leu Leu Trp Leu Thr Ala Thr Ala Ile 340 345 350 Trp Leu Arg Ser Arg Arg Ala Pro Ile Asp His Thr Asn Val Asn Ala 355 360 365 Asp Trp Glu Gly Asn Leu Thr Ala Gly Leu Glu Ala Gly Ala Gln Pro 370 375 380 Leu Ala Asp Gln Arg Pro Pro Val Pro Ala Pro Pro Ala Pro Thr Pro 385 390 395 400 Pro Pro Asn His Arg Ala Pro Gln Phe Gly Val Thr Thr Arg Asn Ala 405 410 415 Pro 4 1251 DNA Mycobacterium tuberculosis 4 gtgaaccttc agttgttcct tttgctcatt gtcgtcgtga cggcattggc gttcgacttc 60 accaacgggt tccacgacac cggaaacgcc atggcgacct cgattgccag cggcgccctg 120 gcaccgcggg tagcggtagc acttcctgcc gtgctgaacc tgatcggtgc gtttttgtcc 180 accgccgtgg cggccacaat cgccaagggt ctgatcgacg cgaatctggt gacgctggag 240 ttggtgttcg ccggcctggt cggcgggatc gtctggaacc tgttgacctg gttgctgggc 300 attccgtcga gttcctcaca tgcgctgatc ggcggcatcg tcggcgccac aattgccgcc 360 gtcggcctgc gtggggtgat ctggagcgga gtggtgtcca aggtgatcgt gccggccgtg 420 gtagccgcgc tgctggccac gctggtcgga gcagtcggca cctggctggt ctaccggacg 480 acgcgcgggg ttgccgaaaa gcgtacggaa cgcggtttcc ggcgcggcca gatcggctcg 540 gcgtcgctgg tctcgctggc gcacggcacc aacgacgcgc agaagacgat gggcgtgatc 600 ttcctggcgt tgatgtccta cggcgcggtc agcacgacgg catcggtgcc gccgctgtgg 660 gtgatcgtga gttgcgccgt ggccatggcc gccggtacct acctgggtgg ctggcgcatc 720 atccgcaccc taggcaaagg gctggtcgag atcaaaccac cgcagggtat ggccgccgag 780 tcgtcatcgg ccgccgtcat tctgttgtcc gcgcacttcg gctatgcgct gtccacaacg 840 caggtcgcga ccgggtccgt gctgggcagc ggcgtcggca agcccggcgc cgaggtgcgc 900 tggggggtag ccggccgcat ggtggtcgcg tggctggtga cgctcccgtt ggccgggctg 960 gtcggggcct tcacctacgg gctggtgcat ttcatcggtg gctaccccgg tgcgatcctc 1020 ggtttcgcac tgttgtggct gaccgccacc gccatctggc tgcggtcgcg cagggcgccg 1080 atcgaccaca ccaacgtcaa cgccgactgg gaaggcaacc tgacggccgg cctggaagcg 1140 ggtgcgcagc cgcttgcgga tcagaggccg ccggtgcctg caccgccggc tccgactccc 1200 ccaccgaacc accgagcacc acagttcggc gtcaccacga ggaacgcccc g 1251 5 231 PRT Mycobacterium tuberculosis 5 Met Thr Ser Gln Thr Gly Val Arg Asp Glu Leu Leu His Ala Gly Val 1 5 10 15 Arg Leu Leu Asp Asp His Gly Pro Asp Ala Leu Gln Thr Arg Lys Val 20 25 30 Ala Ala Ala Ala Gly Thr Ser Thr Met Ala Val Tyr Thr His Phe Gly 35 40 45 Gly Met Arg Gly Leu Ile Ala Ala Ile Ala Glu Glu Gly Leu Arg Gln 50 55 60 Phe Asp Val Ala Leu Thr Val Pro Gln Thr Ala Asp Pro Val Ala Asp 65 70 75 80 Leu Leu Ala Ile Gly Thr Ala Tyr Arg Arg Tyr Ala Ile Glu Arg Pro 85 90 95 His Met Tyr Arg Leu Met Phe Gly Ser Thr Ser Ala His Gly Ile Asn 100 105 110 Val Pro Ala Arg Asp Val Leu Thr Leu Lys Val Ala Glu Ile Glu His 115 120 125 Gln His Pro Ser Phe Ala His Val Val Arg Ala Val His Arg Cys Leu 130 135 140 Leu Ala Gly Arg Phe Ala Thr Ala Leu Gly Ala Asp Asp Asp Thr Ala 145 150 155 160 Ile Val Ala Thr Ala Ala Gln Phe Trp Ser Gln Ile His Gly Phe Val 165 170 175 Met Leu Glu Leu Ala Gly Phe Tyr Gly Asp Arg Gly Ala Ala Val Glu 180 185 190 Pro Val Leu Ala Ala Met Thr Val Asn Leu Leu Val Ala Leu Gly Asp 195 200 205 Ser Pro Glu Arg Ala Gln Cys Ser Leu Arg Ala Glu Gln Thr Gln Lys 210 215 220 Asn Thr Leu Gly Arg Ala Thr 225 230 6 693 DNA Mycobacterium tuberculosis 6 atgacctcgc agaccggtgt tcgcgacgag ctgctgcacg ccggcgtgcg actgctcgac 60 gatcacgggc ccgacgcgct gcagacccgc aaggtggccg ccgcagcagg cacctcgacg 120 atggcggtgt acacccattt cggcgggatg cgcggactga tcgccgccat agccgaagaa 180 gggctacgcc agttcgatgt cgcgctgacg gtcccgcaga ccgccgatcc ggtcgccgac 240 ctgctggcca tcggcaccgc ctaccggcgc tacgccatcg agcgcccgca catgtaccgg 300 ctaatgttcg gcagcaccag cgcacacggc atcaacgtgc cagcgcgcga cgtgttgacc 360 ctcaaggttg ccgagatcga acaccagcac cccagtttcg cgcatgtggt gcgagcggtg 420 caccggtgcc tgctggccgg ccggttcgcg accgcgcttg gagccgacga cgacacggca 480 atagttgcca ccgcggcgca gttttggtca cagatccacg gcttcgtgat gctcgagctg 540 gccggcttct acggcgatcg aggcgcggcc gtcgaaccgg tgctcgccgc gatgacggtg 600 aacctgcttg tcgcgctggg agattcaccc gagcgggcgc agtgttcgct acgggccgag 660 cagacgcaaa agaacacgct gggcagagct act 693 7 217 PRT Mycobacterium tuberculosis 7 Met Ala Arg Lys Gly Ile Leu Gly Thr Lys Leu Gly Met Thr Gln Val 1 5 10 15 Phe Asp Glu Ser Asn Arg Val Val Pro Val Thr Val Val Lys Ala Gly 20 25 30 Pro Asn Val Val Thr Arg Ile Arg Thr Pro Glu Arg Asp Gly Tyr Ser 35 40 45 Ala Val Gln Leu Ala Tyr Gly Glu Ile Ser Pro Arg Lys Val Asn Lys 50 55 60 Pro Leu Thr Gly Gln Tyr Thr Ala Ala Gly Val Asn Pro Arg Arg Tyr 65 70 75 80 Leu Ala Glu Leu Arg Leu Asp Asp Ser Asp Ala Ala Thr Glu Tyr Gln 85 90 95 Val Gly Gln Glu Leu Thr Ala Glu Ile Phe Ala Asp Gly Ser Tyr Val 100 105 110 Asp Val Thr Gly Thr Ser Lys Gly Lys Gly Phe Ala Gly Thr Met Lys 115 120 125 Arg His Gly Phe Arg Gly Gln Gly Ala Ser His Gly Ala Gln Ala Val 130 135 140 His Arg Arg Pro Gly Ser Ile Gly Gly Cys Ala Thr Pro Ala Arg Val 145 150 155 160 Phe Lys Gly Thr Arg Met Ala Gly Arg Met Gly Asn Asp Arg Val Thr 165 170 175 Val Leu Asn Leu Leu Val His Lys Val Asp Ala Glu Asn Gly Val Leu 180 185 190 Leu Ile Lys Gly Ala Val Pro Gly Arg Thr Gly Gly Leu Val Met Val 195 200 205 Arg Ser Ala Ile Lys Arg Gly Glu Lys 210 215 8 651 DNA Mycobacterium tuberculosis 8 atggcacgaa agggcattct cggtaccaag ctgggtatga cgcaggtatt cgacgaaagc 60 aacagagtag taccggtgac cgtggtcaag gccgggccca acgtggtaac ccgcatccgc 120 acgcccgaac gcgacggtta tagcgccgtg cagctggcct atggcgagat cagcccacgc 180 aaggtcaaca agccgctgac aggtcagtac accgccgccg gcgtcaaccc acgccgatac 240 ctggcggagc tgcggctgga cgactcggat gccgcgaccg agtaccaggt tgggcaagag 300 ttgaccgcgg agatcttcgc cgatggcagc tacgtcgatg tgacgggtac ctccaagggc 360 aaaggtttcg ccggcaccat gaagcggcac ggcttccgcg gtcagggcgc cagtcacggt 420 gcccaggcgg tgcaccgccg tccgggctcc atcggcggat gtgccacgcc ggcgcgggtg 480 ttcaagggca cccggatggc cgggcggatg ggcaatgacc gggtgaccgt tcttaacctt 540 ttggtgcata aggtcgatgc cgagaacggc gtgctgctga tcaagggtgc ggttcctggc 600 cgcaccggtg gactggtcat ggtccgcagt gcgatcaaac gaggtgagaa g 651 9 481 PRT Mycobacterium tuberculosis 9 Met Pro Ser Pro Thr Val Thr Ser Pro Gln Val Ala Val Asn Asp Ile 1 5 10 15 Gly Ser Ser Glu Asp Phe Leu Ala Ala Ile Asp Lys Thr Ile Lys Tyr 20 25 30 Phe Asn Asp Gly Asp Ile Val Glu Gly Thr Ile Val Lys Val Asp Arg 35 40 45 Asp Glu Val Leu Leu Asp Ile Gly Tyr Lys Thr Glu Gly Val Ile Pro 50 55 60 Ala Arg Glu Leu Ser Ile Lys His Asp Val Asp Pro Asn Glu Val Val 65 70 75 80 Ser Val Gly Asp Glu Val Glu Ala Leu Val Leu Thr Lys Glu Asp Lys 85 90 95 Glu Gly Arg Leu Ile Leu Ser Lys Lys Arg Ala Gln Tyr Glu Arg Ala 100 105 110 Trp Gly Thr Ile Glu Ala Leu Lys Glu Lys Asp Glu Ala Val Lys Gly 115 120 125 Thr Val Ile Glu Val Val Lys Gly Gly Leu Ile Leu Asp Ile Gly Leu 130 135 140 Arg Gly Phe Leu Pro Ala Ser Leu Val Glu Met Arg Arg Val Arg Asp 145 150 155 160 Leu Gln Pro Tyr Ile Gly Lys Glu Ile Glu Ala Lys Ile Ile Glu Leu 165 170 175 Asp Lys Asn Arg Asn Asn Val Val Leu Ser Arg Arg Ala Trp Leu Glu 180 185 190 Gln Thr Gln Ser Glu Val Arg Ser Glu Phe Leu Asn Asn Leu Gln Lys 195 200 205 Gly Thr Ile Arg Lys Gly Val Val Ser Ser Ile Val Asn Phe Gly Ala 210 215 220 Phe Val Asp Leu Gly Gly Val Asp Gly Leu Val His Val Ser Glu Leu 225 230 235 240 Ser Trp Lys His Ile Asp His Pro Ser Glu Val Val Gln Val Gly Asp 245 250 255 Glu Val Thr Val Glu Val Leu Asp Val Asp Met Asp Arg Glu Arg Val 260 265 270 Ser Leu Ser Leu Lys Ala Thr Gln Glu Asp Pro Trp Arg His Phe Ala 275 280 285 Arg Thr His Ala Ile Gly Gln Ile Val Pro Gly Lys Val Thr Lys Leu 290 295 300 Val Pro Phe Gly Ala Phe Val Arg Val Glu Glu Gly Ile Glu Gly Leu 305 310 315 320 Val His Ile Ser Glu Leu Ala Glu Arg His Val Glu Val Pro Asp Gln 325 330 335 Val Val Ala Val Gly Asp Asp Ala Met Val Lys Val Ile Asp Ile Asp 340 345 350 Leu Glu Arg Arg Arg Ile Ser Leu Ser Leu Lys Gln Ala Asn Glu Asp 355 360 365 Tyr Thr Glu Glu Phe Asp Pro Ala Lys Tyr Gly Met Ala Asp Ser Tyr 370 375 380 Asp Glu Gln Gly Asn Tyr Ile Phe Pro Glu Gly Phe Asp Ala Glu Thr 385 390 395 400 Asn Glu Trp Leu Glu Gly Phe Glu Lys Gln Arg Ala Glu Trp Glu Ala 405 410 415 Arg Tyr Ala Glu Ala Glu Arg Arg His Lys Met His Thr Ala Gln Met 420 425 430 Glu Lys Phe Ala Ala Ala Glu Ala Ala Gly Arg Gly Ala Asp Asp Gln 435 440 445 Ser Ser Ala Ser Ser Ala Pro Ser Glu Lys Thr Ala Gly Gly Ser Leu 450 455 460 Ala Ser Asp Ala Gln Leu Ala Ala Leu Arg Glu Lys Leu Ala Gly Ser 465 470 475 480 Ala 10 1443 DNA Mycobacterium tuberculosis 10 atgccgagtc ccaccgtcac ctcgccgcaa gtagccgtca acgacatagg ctctagcgag 60 gactttctcg ccgcaataga caaaacgatc aagtacttca acgatggcga catcgtcgaa 120 ggcaccatcg tcaaagtgga ccgggacgag gtgctcctcg acatcggcta caagaccgaa 180 ggcgtgatcc ccgcccgcga actgtccatc aagcacgacg tcgaccccaa cgaggtcgtt 240 tccgtcggtg acgaggtcga agccctggtg ctcaccaagg aggacaaaga gggccggctc 300 atcctctcca agaaacgcgc gcagtacgag cgtgcctggg gcaccatcga ggcgctcaag 360 gagaaggacg aggccgtcaa gggcacggtc atcgaggtcg tcaagggtgg cctgatcctc 420 gacatcgggc tgcgcggttt cctgcccgcc tcgctggtgg agatgcgccg ggtgcgcgac 480 ctgcagccct acatcggcaa ggagatcgag gccaagatca tcgagctgga caagaaccgc 540 aacaacgtgg tgctgtcccg tcgcgcctgg ctggagcaga cccagtccga ggtgcgcagc 600 gagttcctga ataacttgca aaaaggcacc atccgaaagg gtgtcgtgtc ctcgatcgtc 660 aacttcggcg cgttcgtcga tctcggcggt gtggacggtc tggtgcatgt ctccgagcta 720 tcgtggaagc acatcgacca cccgtccgag gtggtccagg ttggtgacga ggtcaccgtc 780 gaggtgctcg acgtcgacat ggaccgtgag cgggtttcgt tgtcactcaa ggcgactcag 840 gaagacccgt ggcggcactt cgcccgcact cacgcgatcg ggcagatcgt gccgggcaag 900 gtcaccaagt tggttccgtt cggtgcattc gtccgcgtcg aggagggtat cgagggcctg 960 gtgcacatct ccgagctggc cgagcgtcac gtcgaggtgc ccgatcaggt ggttgccgtc 1020 ggcgacgacg cgatggtcaa ggtcatcgac atcgacctgg agcgccgtcg gatctcgttg 1080 tcgctcaagc aagccaatga ggactacacc gaggagttcg acccggcgaa gtacggcatg 1140 gccgacagtt acgacgagca gggcaactac atcttccccg agggcttcga tgccgaaacc 1200 aacgaatggc ttgagggatt cgaaaagcag cgcgccgaat gggaagctcg gtacgccgag 1260 gccgagcgcc ggcacaagat gcacaccgcg cagatggaga agttcgccgc cgccgaggcg 1320 gctggacgcg gcgcggacga tcagtcgtcg gccagtagcg caccgtcgga aaagaccgcg 1380 ggtggatcac tggccagcga cgcccagctg gcggccctgc gggaaaaact cgccggcagc 1440 gct 1443 11 380 PRT Mycobacterium tuberculosis 11 Leu Asp Thr Val Leu Gly Leu Ser Ile Thr Pro Thr Thr Leu Gly Trp 1 5 10 15 Val Leu Ala Glu Gly His Gly Ala Asp Gly Ala Ile Leu Asp Arg Asn 20 25 30 Glu Leu Glu Leu His Ser Gly Arg Asn Ala Gln Ala Ile His Thr Ala 35 40 45 Glu Gln Leu Ala Ala Glu Val Leu Leu Ala His Glu Val Ala Ala Ala 50 55 60 Gly Asp His Arg Leu Arg Val Ile Gly Val Thr Trp Asn Ala Glu Ala 65 70 75 80 Ser Ala Gln Ala Ala Leu Leu Val Glu Ser Leu Thr Gly Ala Gly Phe 85 90 95 Asp Asn Val Val Pro Val Arg Arg Leu Arg Ala Ile Glu Thr Leu Ala 100 105 110 Gln Ala Ile Ala Pro Val Ile Gly Tyr Glu Gln Ile Ala Val Cys Val 115 120 125 Leu Glu His Glu Ser Ala Thr Val Val Met Val Asp Thr His Asp Gly 130 135 140 Lys Thr Gln Ile Ala Val Lys His Val Cys Arg Gly Leu Ser Gly Leu 145 150 155 160 Thr Ser Trp Leu Thr Gly Met Phe Gly Arg Asp Ala Trp Arg Pro Ala 165 170 175 Gly Val Val Val Val Gly Ser Asp Ser Glu Val Ser Glu Phe Ser Trp 180 185 190 Gln Leu Glu Arg Val Leu Pro Val Pro Val Phe Ala Gln Thr Met Ala 195 200 205 Gln Val Thr Val Ala Arg Gly Ala Ala Leu Ala Ala Ala Gln Ser Thr 210 215 220 Glu Phe Thr Asp Ala Gln Leu Val Ala Asp Ser Val Ser Gln Pro Thr 225 230 235 240 Val Ala Pro Arg Arg Ser Arg His Tyr Ala Gly Ala Ala Ala Ala Leu 245 250 255 Ala Ala Ala Ala Val Thr Phe Val Ala Ser Leu Ser Leu Ala Val Gly 260 265 270 Ile Gln Leu Ala Pro His Asn Asp Thr Gly Thr Ala Lys His Gly Ala 275 280 285 His Lys Pro Thr Pro Arg Ile Ala Lys Ala Val Ala Pro Ala Val Pro 290 295 300 Pro Pro Pro Thr Val Thr Pro Pro Val Pro Ala Arg Ala Pro Arg Pro 305 310 315 320 Ala Ala Gln His Glu Pro Pro Ala Arg Val Thr Ser Gly Glu Ala Leu 325 330 335 Thr Glu Pro Asn Pro Pro Glu Glu Gln Pro Asn Ala Ser Ala Pro Gln 340 345 350 Gln Asp Arg Asn Asp Ser Gln Pro Ile Thr Arg Val Leu Glu His Ile 355 360 365 Pro Gly Ala Tyr Gly Asp Ser Ala Pro Pro Ala Glu 370 375 380 12 1140 DNA Mycobacterium tuberculosis 12 ttggacacgg tacttgggct ctcgataacg cctaccaccc tggggtgggt cctcgctgaa 60 ggacacggcg cagacggcgc catcttggac cgcaacgaat tggagctaca tagcggtcgt 120 aacgcgcagg ccatacatac cgcagagcag ctggcggcgg aagttctgct cgcccatgaa 180 gtggccgctg caggcgatca tcggttgcgc gtcatcggag tgacctggaa cgccgaagct 240 tcggctcagg cggcgctgct ggtagagtcg ctgaccggtg caggtttcga caatgtggtg 300 ccggttcggc ggctacgtgc catcgagaca ctggcgcagg ctatcgcacc cgttatcggc 360 tacgagcaaa tcgcggtatg cgttcttgag catgagtcgg cgaccgtcgt catggtcgac 420 acccacgacg gaaagacgca gatcgccgtc aagcatgtgt gccgcggatt atcaggactg 480 acctcctggc tgaccggcat gtttggtcgc gatgcctggc gcccggccgg cgtggtcgtg 540 gtcggctcgg atagcgaggt cagcgaattc tcgtggcagc tcgaaagggt cctgccggtg 600 ccggtctttg cgcaaacgat ggcgcaggtt acggtcgcgc ggggtgcggc cctggcggcg 660 gcccagagca ccgagttcac cgatgcgcag ctagtggccg acagcgtcag ccaaccaacg 720 gtcgcgccca ggcgatcccg gcactacgcc ggggcggcgg cagcgttggc cgccgcggcc 780 gtgaccttcg tggcttcgct gtccctagcg gtgggcatcc agctggctcc gcacaacgat 840 accgggacgg cgaagcacgg agcgcacaag ccgacgccac gtatcgcaaa ggccgtggcg 900 ccggcggtgc cgcctccgcc gacggtcacg ccaccagtcc ctgctcgggc accccggccg 960 gctgcgcagc acgaaccacc cgctcgcgtc acctccggcg aagcgctcac ggagccgaac 1020 ccgcctgagg agcaaccgaa tgcttctgcg ccgcaacagg atcggaatga cagccagccg 1080 atcactcgag tgctagagca catacccggc gcttacggtg actcggcacc cccagctgag 1140 13 118 PRT Mycobacterium tuberculosis 13 Met Leu Ile Ile Ala Leu Val Leu Ala Leu Ile Gly Leu Leu Ala Leu 1 5 10 15 Val Phe Ala Val Val Thr Ser Asn Gln Leu Val Ala Trp Val Cys Ile 20 25 30 Gly Ala Ser Val Leu Gly Val Ala Leu Leu Ile Val Asp Ala Leu Arg 35 40 45 Glu Arg Gln Gln Gly Gly Ala Asp Glu Ala Asp Gly Ala Gly Glu Thr 50 55 60 Gly Val Ala Glu Glu Ala Asp Val Asp Tyr Pro Glu Glu Ala Pro Glu 65 70 75 80 Glu Ser Gln Ala Val Asp Ala Gly Val Ile Gly Ser Glu Glu Pro Ser 85 90 95 Glu Glu Ala Ser Glu Ala Thr Glu Glu Ser Ala Val Ser Ala Asp Arg 100 105 110 Ser Asp Asp Ser Ala Lys 115 14 354 DNA Mycobacterium tuberculosis 14 atgctgatca ttgcgctggt cttggccctg attgggctcc tggccttggt gttcgcggtg 60 gtcaccagca accagctagt ggcctgggta tgcatcgggg ccagcgtgct gggtgtggcg 120 ttgctgatcg tcgatgcgtt gcgagaacgc cagcaaggtg gcgcggacga agctgatggg 180 gctggggaaa cgggtgtcgc ggaggaagcc gacgtcgact acccggagga agcccccgag 240 gagagccaag ccgtcgacgc cggtgtcatc ggcagtgagg agccatcgga ggaggccagc 300 gaagcgaccg aggagtcggc ggtatcggcg gaccgaagcg acgacagcgc caag 354 15 143 PRT Mycobacterium tuberculosis 15 Met Thr Ala Pro Ala Ser Leu Pro Ala Pro Leu Ala Glu Val Val Ser 1 5 10 15 Asp Phe Ala Glu Val Gln Gly Gln Asp Lys Leu Arg Leu Leu Leu Glu 20 25 30 Phe Ala Asn Glu Leu Pro Ala Leu Pro Ser His Leu Ala Glu Ser Ala 35 40 45 Met Glu Pro Val Pro Glu Cys Gln Ser Pro Leu Phe Leu His Val Asp 50 55 60 Ala Ser Asp Pro Asn Arg Val Arg Leu His Phe Ser Ala Pro Ala Glu 65 70 75 80 Ala Pro Thr Thr Arg Gly Phe Ala Ser Ile Leu Ala Ala Gly Leu Asp 85 90 95 Glu Gln Pro Ala Ala Asp Ile Leu Ala Val Pro Glu Asp Phe Tyr Thr 100 105 110 Glu Leu Gly Leu Ala Ala Leu Ile Ser Pro Leu Arg Leu Arg Gly Met 115 120 125 Ser Ala Met Leu Ala Arg Ile Lys Arg Arg Leu Arg Glu Ala Asp 130 135 140 16 429 DNA Mycobacterium tuberculosis 16 atgaccgcgc ccgcgagcct gcccgcgccg ctagcagagg tggtatccga cttcgccgaa 60 gtccagggtc aagacaagct gaggctgttg ctggaattcg ccaacgagct gccggcgctt 120 ccgtcgcacc tggccgagtc cgctatggag ccggtccccg agtgccagtc tccgctgttt 180 ttgcacgtcg acgcgagtga ccccaaccgg gtgcgcctgc atttcagcgc gccggccgaa 240 gcgccaacca cgcgcgggtt cgcctcgatc ctggccgccg gcctagacga gcaaccggcc 300 gccgacatct tggcggtgcc cgaggatttc tacaccgagc tgggtctggc tgccttgatc 360 agcccactgc ggttgcgggg aatgtcggcg atgctggccc ggatcaagcg ccggctgcgc 420 gaagcggac 429 17 201 PRT Mycobacterium tuberculosis 17 Met Ala Arg Tyr Thr Gly Pro Val Thr Arg Lys Ser Arg Arg Leu Arg 1 5 10 15 Thr Asp Leu Val Gly Gly Asp Gln Ala Phe Glu Lys Arg Pro Tyr Pro 20 25 30 Pro Gly Gln His Gly Arg Ala Arg Ile Lys Glu Ser Glu Tyr Leu Leu 35 40 45 Gln Leu Gln Glu Lys Gln Lys Ala Arg Phe Thr Tyr Gly Val Met Glu 50 55 60 Lys Gln Phe Arg Arg Tyr Tyr Glu Glu Ala Val Arg Gln Pro Gly Lys 65 70 75 80 Thr Gly Glu Glu Leu Leu Lys Ile Leu Glu Ser Arg Leu Asp Asn Val 85 90 95 Ile Tyr Arg Ala Gly Leu Ala Arg Thr Arg Arg Met Ala Arg Gln Leu 100 105 110 Val Ser His Gly His Phe Asn Val Asn Gly Val His Val Asn Val Pro 115 120 125 Ser Tyr Arg Val Ser Gln Tyr Asp Ile Val Asp Val Arg Asp Lys Ser 130 135 140 Leu Asn Thr Val Pro Phe Gln Ile Ala Arg Glu Thr Ala Gly Glu Arg 145 150 155 160 Pro Ile Pro Ser Trp Leu Gln Val Val Gly Glu Arg Gln Arg Val Leu 165 170 175 Ile His Gln Leu Pro Glu Arg Ala Gln Ile Asp Val Pro Leu Thr Glu 180 185 190 Gln Leu Ile Val Glu Tyr Tyr Ser Lys 195 200 18 603 DNA Mycobacterium tuberculosis 18 atggctcgtt acaccggacc cgtcacccgc aaatcacggc ggttgcgcac cgacctcgtc 60 ggtggcgacc aggccttcga gaagcgtccc tacccgcccg gccaacacgg tcgcgcgcgg 120 atcaaggaaa gcgaatatct gcttcagctg caggagaagc agaaggcccg tttcacatac 180 ggcgtaatgg aaaagcagtt ccgccgctac tacgaagagg ccgtgcggca gcccggcaag 240 acgggtgaag aactgctgaa gatcctcgaa agccggctgg acaacgtcat ctaccgtgcc 300 gggctggcgc gcacccggcg gatggctcgc cagctggtca gccacgggca tttcaacgtc 360 aacggcgtgc acgtcaacgt ccccagttac cgggtgtcgc agtacgacat cgtcgacgtg 420 cgggacaagt ccctgaacac ggtgccgttc cagattgccc gggagacggc gggcgagcgt 480 ccgatcccga gctggctgca agtggtgggg gagcggcaac gcgtcctgat ccaccagcta 540 cccgagcgcg cgcagatcga cgtcccactc accgagcagc tgatcgtcga gtactactca 600 aag 603 19 393 PRT Mycobacterium tuberculosis 19 Val Val Asp Phe Gly Ala Leu Pro Pro Glu Ile Asn Ser Ala Arg Met 1 5 10 15 Tyr Ala Gly Pro Gly Ser Ala Ser Leu Val Ala Ala Ala Lys Met Trp 20 25 30 Asp Ser Val Ala Ser Asp Leu Phe Ser Ala Ala Ser Ala Phe Gln Ser 35 40 45 Val Val Trp Gly Leu Thr Val Gly Ser Trp Ile Gly Ser Ser Ala Gly 50 55 60 Leu Met Ala Ala Ala Ala Ser Pro Tyr Val Ala Trp Met Ser Val Thr 65 70 75 80 Ala Gly Gln Ala Gln Leu Thr Ala Ala Gln Val Arg Val Ala Ala Ala 85 90 95 Ala Tyr Glu Thr Ala Tyr Arg Leu Thr Val Pro Pro Pro Val Ile Ala 100 105 110 Glu Asn Arg Thr Glu Leu Met Thr Leu Thr Ala Thr Asn Leu Leu Gly 115 120 125 Gln Asn Thr Pro Ala Ile Glu Ala Asn Gln Ala Ala Tyr Ser Gln Met 130 135 140 Trp Gly Gln Asp Ala Glu Ala Met Tyr Gly Tyr Ala Ala Thr Ala Ala 145 150 155 160 Thr Ala Thr Glu Ala Leu Leu Pro Phe Glu Asp Ala Pro Leu Ile Thr 165 170 175 Asn Pro Gly Gly Leu Leu Glu Gln Ala Val Ala Val Glu Glu Ala Ile 180 185 190 Asp Thr Ala Ala Ala Asn Gln Leu Met Asn Asn Val Pro Gln Ala Leu 195 200 205 Gln Gln Leu Ala Gln Pro Ala Gln Gly Val Val Pro Ser Ser Lys Leu 210 215 220 Gly Gly Leu Trp Thr Ala Val Ser Pro His Leu Ser Pro Leu Ser Asn 225 230 235 240 Val Ser Ser Ile Ala Asn Asn His Met Ser Met Met Gly Thr Gly Val 245 250 255 Ser Met Thr Asn Thr Leu His Ser Met Leu Lys Gly Leu Ala Pro Ala 260 265 270 Ala Ala Gln Ala Val Glu Thr Ala Ala Glu Asn Gly Val Trp Ala Met 275 280 285 Ser Ser Leu Gly Ser Gln Leu Gly Ser Ser Leu Gly Ser Ser Gly Leu 290 295 300 Gly Ala Gly Val Ala Ala Asn Leu Gly Arg Ala Ala Ser Val Gly Ser 305 310 315 320 Leu Ser Val Pro Pro Ala Trp Ala Ala Ala Asn Gln Ala Val Thr Pro 325 330 335 Ala Ala Arg Ala Leu Pro Leu Thr Ser Leu Thr Ser Ala Ala Gln Thr 340 345 350 Ala Pro Gly His Met Leu Gly Gly Leu Pro Leu Gly His Ser Val Asn 355 360 365 Ala Gly Ser Gly Ile Asn Asn Ala Leu Arg Val Pro Ala Arg Ala Tyr 370 375 380 Ala Ile Pro Arg Thr Pro Ala Ala Gly 385 390 20 1179 DNA Mycobacterium tuberculosis 20 gtggtggatt tcggggcgtt accaccggag atcaactccg cgaggatgta cgccggcccg 60 ggttcggcct cgctggtggc cgccgcgaag atgtgggaca gcgtggcgag tgacctgttt 120 tcggccgcgt cggcgtttca gtcggtggtc tggggtctga cggtggggtc gtggataggt 180 tcgtcggcgg gtctgatggc ggcggcggcc tcgccgtatg tggcgtggat gagcgtcacc 240 gcggggcagg cccagctgac cgccgcccag gtccgggttg ctgcggcggc ctacgagaca 300 gcgtataggc tgacggtgcc cccgccggtg atcgccgaga accgtaccga actgatgacg 360 ctgaccgcga ccaacctctt ggggcaaaac acgccggcga tcgaggccaa tcaggccgca 420 tacagccaga tgtggggcca agacgcggag gcgatgtatg gctacgccgc cacggcggcg 480 acggcgaccg aggcgttgct gccgttcgag gacgccccac tgatcaccaa ccccggcggg 540 ctccttgagc aggccgtcgc ggtcgaggag gccatcgaca ccgccgcggc gaaccagttg 600 atgaacaatg tgccccaagc gctgcaacag ctggcccagc cagcgcaggg cgtcgtacct 660 tcttccaagc tgggtgggct gtggacggcg gtctcgccgc atctgtcgcc gctcagcaac 720 gtcagttcga tagccaacaa ccacatgtcg atgatgggca cgggtgtgtc gatgaccaac 780 accttgcact cgatgttgaa gggcttagct ccggcggcgg ctcaggccgt ggaaaccgcg 840 gcggaaaacg gggtctgggc gatgagctcg ctgggcagcc agctgggttc gtcgctgggt 900 tcttcgggtc tgggcgctgg ggtggccgcc aacttgggtc gggcggcctc ggtcggttcg 960 ttgtcggtgc cgccagcatg ggccgcggcc aaccaggcgg tcaccccggc ggcgcgggcg 1020 ctgccgctga ccagcctgac cagcgccgcc caaaccgccc ccggacacat gctgggcggg 1080 ctaccgctgg ggcactcggt caacgccggc agcggtatca acaatgcgct gcgggtgccg 1140 gcacgggcct acgcgatacc ccgcacaccg gccgccgga 1179 21 565 PRT Mycobacterium tuberculosis 21 Met Thr Arg Gly Arg Lys Pro Arg Pro Gly Arg Ile Val Phe Val Gly 1 5 10 15 Ser Gly Pro Gly Asp Pro Gly Leu Leu Thr Thr Arg Ala Ala Ala Val 20 25 30 Leu Ala Asn Ala Ala Leu Val Phe Thr Asp Pro Asp Val Pro Glu Pro 35 40 45 Val Val Ala Leu Ile Gly Thr Asp Leu Pro Pro Val Ser Gly Pro Ala 50 55 60 Pro Ala Glu Pro Val Ala Gly Asn Gly Asp Ala Ala Gly Gly Gly Ser 65 70 75 80 Ala Gln Glu His Gly Arg Ala Ala Ser Ala Val Val Ser Gly Gly Pro 85 90 95 Asp Ile Arg Pro Ala Leu Gly Asp Pro Ala Asp Val Ala Lys Thr Leu 100 105 110 Thr Ala Glu Ala Arg Ser Gly Val Asp Val Val Arg Leu Val Ala Gly 115 120 125 Asp Pro Leu Thr Val Asp Ala Val Ile Ser Glu Val Asn Ala Val Ala 130 135 140 Arg Thr His Leu His Ile Glu Ile Val Pro Gly Leu Ala Ala Ser Ser 145 150 155 160 Ala Val Pro Thr Tyr Ala Gly Leu Pro Leu Gly Ser Ser His Thr Val 165 170 175 Ala Asp Val Arg Ile Asp Pro Glu Asn Thr Asp Trp Asp Ala Leu Ala 180 185 190 Ala Ala Pro Gly Pro Leu Ile Leu Gln Ala Thr Ala Ser His Leu Ala 195 200 205 Glu Ser Ala Arg Ser Leu Ile Asp His Gln Leu Ala Glu Ser Thr Pro 210 215 220 Cys Val Val Thr Ala His Gly Thr Thr Cys Gln Gln Arg Ser Val Glu 225 230 235 240 Thr Thr Leu Gln Gly Leu Thr Asp Pro Ala Val Leu Gly Ala Thr Asp 245 250 255 Pro Ala Cys Ser Ala Asn Gly Arg Asp Ser Gln Ala Gly Pro Leu Ile 260 265 270 Val Thr Ile Gly Lys Thr Val Thr Ser Arg Ala Lys Leu Asn Trp Trp 275 280 285 Glu Ser Arg Ala Leu Tyr Gly Trp Thr Val Leu Val Pro Arg Thr Lys 290 295 300 Asp Gln Ala Gly Glu Met Ser Glu Arg Leu Thr Ser Tyr Gly Ala Leu 305 310 315 320 Pro Val Glu Val Pro Thr Ile Ala Val Glu Pro Pro Arg Ser Pro Ala 325 330 335 Gln Met Glu Arg Ala Val Lys Gly Leu Val Asp Gly Arg Phe Gln Trp 340 345 350 Ile Val Phe Thr Ser Thr Asn Ala Val Arg Ala Val Trp Glu Lys Phe 355 360 365 Gly Glu Phe Gly Leu Asp Ala Arg Ala Phe Ser Gly Val Lys Ile Ala 370 375 380 Cys Val Gly Glu Ser Thr Ala Asp Arg Val Arg Ala Phe Gly Ile Ser 385 390 395 400 Pro Glu Leu Val Pro Ser Gly Glu Gln Ser Ser Leu Gly Leu Leu Asp 405 410 415 Asp Phe Pro Pro Tyr Asp Ser Val Phe Asp Pro Val Asn Arg Val Leu 420 425 430 Leu Pro Arg Ala Asp Ile Ala Thr Glu Thr Leu Ala Glu Gly Leu Arg 435 440 445 Glu Arg Gly Trp Glu Ile Glu Asp Val Thr Ala Tyr Arg Thr Val Arg 450 455 460 Ala Ala Pro Pro Pro Ala Thr Thr Arg Glu Met Ile Lys Thr Gly Gly 465 470 475 480 Phe Asp Ala Val Cys Phe Thr Ser Ser Ser Thr Val Arg Asn Leu Val 485 490 495 Gly Ile Ala Gly Lys Pro His Ala Arg Thr Ile Ile Ala Cys Ile Gly 500 505 510 Pro Lys Thr Ala Glu Thr Ala Ala Glu Phe Gly Leu Arg Val Asp Val 515 520 525 Gln Pro Asp Thr Ala Ala Ile Gly Pro Leu Val Asp Ala Leu Ala Glu 530 535 540 His Ala Ala Arg Leu Arg Ala Glu Gly Ala Leu Pro Pro Pro Arg Lys 545 550 555 560 Lys Ser Arg Arg Arg 565 22 1695 DNA Mycobacterium tuberculosis 22 atgacgcgag ggcgtaagcc gagaccgggc cgcatcgttt tcgtgggctc cggtccgggc 60 gaccccggct tgcttacgac acgggctgcc gcggtgctgg ccaacgccgc gctggtgttc 120 accgatcccg acgtaccgga gccggtggtg gcgctgatcg gcacggatct gccccccgtg 180 tccggcccgg cgcccgccga gccggttgcc gggaacggcg atgcggccgg cggaggaagt 240 gcgcaggaac acggccgggc cgcgtccgcg gtagtctccg gtggtcctga catccgcccg 300 gcgctgggcg atcccgccga tgtggccaag acgctgaccg ccgaggcccg ttcgggtgtc 360 gacgtggtgc ggctggtggc gggcgatccg ctcacggtgg atgcggtaat cagcgaggtg 420 aacgccgtcg cacgcaccca cctgcacatc gaaatcgtgc ccggcctggc cgccagcagc 480 gcggtcccga cctatgccgg gttgccgctg ggttcgtcgc acaccgtcgc cgacgtgcgt 540 atcgaccccg aaaacaccga ctgggacgcg ctggctgccg cacccgggcc gctgatcctg 600 caggccaccg catcgcatct agccgaatcg gcccgcagcc tgatcgatca ccagctggcc 660 gagtccactc cgtgcgtggt gaccgcacac ggcaccacct gtcagcagcg ttcggtcgag 720 accacacttc agggattgac cgacccggcc gtcctgggcg ctaccgaccc cgcgtgctcc 780 gcaaacggga gggactccca ggccggaccg ctgatagtga ccatcggcaa gacggtgacc 840 agtcgggcaa agctgaactg gtgggagagc cgcgccctct acggctggac ggtgttggtg 900 ccgcgcacca aggaccaggc cggcgagatg agcgagcggc tcacgtcgta cggcgcgctg 960 ccggtggagg tgccgaccat cgccgtcgag ccgccgcgca gccccgcgca gatggagcgc 1020 gccgtcaagg gcctggtcga tggccgattc cagtggatcg tgttcacctc caccaacgcg 1080 gtgcgtgcgg tgtgggagaa gttcggcgag ttcggtctgg atgcccgcgc gttctccggg 1140 gtgaagatcg cctgtgtcgg cgagtcgacg gccgaccggg tgcgcgcctt cggaatcagt 1200 cccgagctgg tgccctccgg ggagcagtcc tcgcttggct tgctagacga cttcccgccc 1260 tacgacagcg ttttcgaccc ggtgaaccgg gttttgctgc cgcgcgccga catcgccacc 1320 gaaacgctgg ccgagggact gcgagagcgt ggctgggaga tcgaggacgt caccgcctac 1380 cggaccgtgc gggccgcgcc gccgccggcc actacccggg aaatgatcaa gacgggcggg 1440 tttgacgcgg tatgtttcac ctccagctcg acggtgcgaa acctggtcgg catcgccggc 1500 aagccgcacg cgcggacgat catcgcctgc atagggccaa agaccgccga gaccgcagcc 1560 gagttcggct tgcgggtcga tgtccagccg gacaccgccg ccatcggccc gctggtcgat 1620 gcgctggccg agcatgccgc ccggttgcgc gctgagggtg cgctgccccc gccgcgcaag 1680 aagagccgca ggcgc 1695 23 388 PRT Mycobacterium tuberculosis 23 Met Ser Glu Asp Arg Thr Gly His Gln Gly Ile Ser Gly Pro Ala Thr 1 5 10 15 Arg Ala Ile His Ala Gly Tyr Arg Pro Asp Pro Ala Thr Gly Ala Val 20 25 30 Asn Val Pro Ile Tyr Ala Ser Ser Thr Phe Ala Gln Asp Gly Val Gly 35 40 45 Gly Leu Arg Gly Gly Phe Glu Tyr Ala Arg Thr Gly Asn Pro Thr Arg 50 55 60 Ala Ala Leu Glu Ala Ser Leu Ala Ala Val Glu Glu Gly Ala Phe Ala 65 70 75 80 Arg Ala Phe Ser Ser Gly Met Ala Ala Thr Asp Cys Ala Leu Arg Ala 85 90 95 Met Leu Arg Pro Gly Asp His Val Val Ile Pro Asp Asp Ala Tyr Gly 100 105 110 Gly Thr Phe Arg Leu Ile Asp Lys Val Phe Thr Arg Trp Asp Val Gln 115 120 125 Tyr Thr Pro Val Arg Leu Ala Asp Leu Asp Ala Val Gly Ala Ala Ile 130 135 140 Thr Pro Arg Thr Arg Leu Ile Trp Val Glu Thr Pro Thr Asn Pro Leu 145 150 155 160 Leu Ser Ile Ala Asp Ile Thr Ala Ile Ala Glu Leu Gly Thr Asp Arg 165 170 175 Ser Ala Lys Val Leu Val Asp Asn Thr Phe Ala Ser Pro Ala Leu Gln 180 185 190 Gln Pro Leu Arg Leu Gly Ala Asp Val Val Leu His Ser Thr Thr Lys 195 200 205 Tyr Ile Gly Gly His Ser Asp Val Val Gly Gly Ala Leu Val Thr Asn 210 215 220 Asp Glu Glu Leu Asp Glu Glu Phe Ala Phe Leu Gln Asn Gly Ala Gly 225 230 235 240 Ala Val Pro Gly Pro Phe Asp Ala Tyr Leu Thr Met Arg Gly Leu Lys 245 250 255 Thr Leu Val Leu Arg Met Gln Arg His Ser Glu Asn Ala Cys Ala Val 260 265 270 Ala Glu Phe Leu Ala Asp His Pro Ser Val Ser Ser Val Leu Tyr Pro 275 280 285 Gly Leu Pro Ser His Pro Gly His Glu Ile Ala Ala Arg Gln Met Arg 290 295 300 Gly Phe Gly Gly Met Val Ser Val Arg Met Arg Ala Gly Arg Arg Ala 305 310 315 320 Ala Gln Asp Leu Cys Ala Lys Thr Arg Val Phe Ile Leu Ala Glu Ser 325 330 335 Leu Gly Gly Val Glu Ser Leu Ile Glu His Pro Ser Ala Met Thr His 340 345 350 Ala Ser Thr Ala Gly Ser Gln Leu Glu Val Pro Asp Asp Leu Val Arg 355 360 365 Leu Ser Val Gly Ile Glu Asp Ile Ala Asp Leu Leu Gly Asp Leu Glu 370 375 380 Gln Ala Leu Gly 385 24 1164 DNA Mycobacterium tuberculosis 24 atgagcgaag accgcacggg acaccaggga atcagcggac cggccacccg cgccatccac 60 gctggctacc gcccggatcc ggcgaccggg gcggtgaacg tgccgatcta cgccagcagc 120 accttcgccc aagacggcgt cggcggtctg cgtggcggtt tcgaatacgc acgcaccggc 180 aaccccaccc gggccgcatt ggaggcctcg ctggcggcag tcgaggaggg tgctttcgcg 240 cgggcattca gttccgggat ggccgcgacc gactgcgccc tgcgggcgat gttacggccc 300 ggagaccacg tcgtcattcc cgatgacgcc tacggcggca cattccggtt gatagacaag 360 gtgttcaccc ggtgggatgt ccagtacacg ccggtgcggc ttgccgatct ggatgcggtg 420 ggtgccgcga ttactccgcg cacccggctg atttgggtgg agacgcccac caatccgcta 480 ctgtcgatcg ccgatatcac ggccattgcc gagctgggca cagacagatc ggcaaaagta 540 ttggtggaca atacctttgc ctcacccgcg ttgcagcagc cgttgcggct gggcgccgat 600 gtggtgttgc actcgactac caagtacatc ggcggccatt ccgacgtggt gggaggtgcg 660 ctggtcacca acgacgaaga gctggacgag gagttcgctt tcttgcagaa cggcgccggc 720 gcggtgcccg gaccattcga cgcctacctg accatgcgcg gcctgaagac cttggtgctg 780 cggatgcagc ggcacagtga aaatgcctgt gcggtagcgg aattcctcgc tgatcatccg 840 tcggtgagtt ctgtgttgta tccgggtttg cccagtcatc ccgggcatga gattgccgcg 900 cgacagatgc gcggcttcgg cggcatggtt tcggtgcgga tgcgggccgg tcggcgtgcg 960 gcgcaggacc tgtgtgccaa gacccgcgtc ttcatcctgg ccgagtcgct gggtggggtg 1020 gagtcgctga tcgaacatcc cagcgccatg acccatgcgt cgacggccgg ttcgcaattg 1080 gaggtgcccg acgatctggt gcggctttcg gtcggtatcg aagacattgc cgacctgctc 1140 ggcgatctcg aacaggccct gggt 1164 25 944 PRT Mycobacterium tuberculosis 25 Val Phe Ala Trp Trp Gly Arg Thr Val Tyr Arg Tyr Arg Phe Ile Val 1 5 10 15 Ile Gly Val Met Val Ala Leu Cys Leu Gly Gly Gly Val Phe Gly Leu 20 25 30 Ser Leu Gly Lys His Val Thr Gln Ser Gly Phe Tyr Asp Asp Gly Ser 35 40 45 Gln Ser Val Gln Ala Ser Val Leu Gly Asp Gln Val Tyr Gly Arg Asp 50 55 60 Arg Ser Gly His Ile Val Ala Ile Phe Gln Ala Pro Ala Gly Lys Thr 65 70 75 80 Val Asp Asp Pro Ala Trp Ser Lys Lys Val Val Asp Glu Leu Asn Arg 85 90 95 Phe Gln Gln Asp His Pro Asp Gln Val Leu Gly Trp Ala Gly Tyr Leu 100 105 110 Arg Ala Ser Gln Ala Thr Gly Met Ala Thr Ala Asp Lys Lys Tyr Thr 115 120 125 Phe Val Ser Ile Pro Leu Lys Gly Asp Asp Asp Asp Thr Ile Leu Asn 130 135 140 Asn Tyr Lys Ala Ile Ala Pro Asp Leu Gln Arg Leu Asp Gly Gly Thr 145 150 155 160 Val Lys Leu Ala Gly Leu Gln Pro Val Ala Glu Ala Leu Thr Gly Thr 165 170 175 Ile Ala Thr Asp Gln Arg Arg Met Glu Val Leu Ala Leu Pro Leu Val 180 185 190 Ala Val Val Leu Phe Phe Val Phe Gly Gly Val Ile Ala Ala Gly Leu 195 200 205 Pro Val Met Val Gly Gly Leu Cys Ile Ala Gly Ala Leu Gly Ile Met 210 215 220 Arg Phe Leu Ala Ile Phe Gly Pro Val His Tyr Phe Ala Gln Pro Val 225 230 235 240 Val Ser Leu Ile Gly Leu Gly Ile Ala Ile Asp Tyr Gly Leu Phe Ile 245 250 255 Val Ser Arg Phe Arg Glu Glu Ile Ala Glu Gly Tyr Asp Thr Glu Thr 260 265 270 Ala Val Arg Arg Thr Val Ile Thr Ala Gly Arg Thr Val Thr Phe Ser 275 280 285 Ala Val Leu Ile Val Ala Ser Ala Ile Gly Leu Leu Leu Phe Pro Gln 290 295 300 Gly Phe Leu Lys Ser Leu Thr Tyr Ala Thr Ile Ala Ser Val Met Leu 305 310 315 320 Ser Ala Ile Leu Ser Ile Thr Val Leu Pro Ala Cys Leu Gly Ile Leu 325 330 335 Gly Lys His Val Asp Ala Leu Gly Val Arg Thr Leu Phe Arg Val Pro 340 345 350 Phe Leu Ala Asn Trp Lys Ile Ser Ala Ala Tyr Leu Asn Trp Leu Ala 355 360 365 Asp Arg Leu Gln Arg Thr Lys Thr Arg Glu Glu Val Glu Ala Gly Phe 370 375 380 Trp Gly Lys Leu Val Asn Arg Val Met Lys Arg Pro Val Leu Phe Ala 385 390 395 400 Ala Pro Ile Val Ile Ile Met Ile Leu Leu Ile Ile Pro Val Gly Lys 405 410 415 Leu Ser Leu Gly Gly Ile Ser Glu Lys Tyr Leu Pro Pro Thr Asn Ser 420 425 430 Val Arg Gln Ala Gln Glu Glu Phe Asp Lys Leu Phe Pro Gly Tyr Arg 435 440 445 Thr Asn Pro Leu Thr Leu Val Ile Gln Thr Ser Asn His Gln Pro Val 450 455 460 Thr Asp Ala Gln Ile Ala Asp Ile Arg Ser Lys Ala Met Ala Ile Gly 465 470 475 480 Gly Phe Ile Glu Pro Asp Asn Asp Pro Ala Asn Met Trp Gln Glu Arg 485 490 495 Ala Tyr Ala Val Gly Ala Ser Lys Asp Pro Ser Val Arg Val Leu Gln 500 505 510 Asn Gly Leu Ile Asn Pro Ala Asp Ala Ser Lys Lys Leu Thr Glu Leu 515 520 525 Arg Ala Ile Thr Pro Pro Lys Gly Ile Thr Val Leu Val Gly Gly Thr 530 535 540 Pro Ala Leu Glu Leu Asp Ser Ile His Gly Leu Phe Ala Lys Met Pro 545 550 555 560 Leu Met Val Val Ile Leu Leu Thr Thr Thr Ile Val Leu Met Phe Leu 565 570 575 Ala Phe Gly Ser Val Val Leu Pro Ile Lys Ala Thr Leu Met Ser Ala 580 585 590 Leu Thr Leu Gly Ser Thr Met Gly Ile Leu Thr Trp Ile Phe Val Asp 595 600 605 Gly His Phe Ser Lys Trp Leu Asn Phe Thr Pro Thr Pro Leu Thr Ala 610 615 620 Pro Val Ile Gly Leu Ile Ile Ala Leu Val Phe Gly Leu Ser Thr Asp 625 630 635 640 Tyr Glu Val Phe Leu Val Ser Arg Met Val Glu Ala Arg Glu Arg Gly 645 650 655 Met Ser Thr Gln Glu Ala Ile Arg Ile Gly Thr Ala Ala Thr Gly Arg 660 665 670 Ile Ile Thr Ala Ala Ala Leu Ile Val Ala Val Val Ala Gly Ala Phe 675 680 685 Val Phe Ser Asp Leu Val Met Met Lys Tyr Leu Ala Phe Gly Leu Met 690 695 700 Ala Ala Leu Leu Leu Asp Ala Thr Val Val Arg Met Phe Leu Val Pro 705 710 715 720 Ser Val Met Lys Leu Leu Gly Asp Asp Cys Trp Trp Ala Pro Arg Trp 725 730 735 Ala Arg Arg Leu Gln Thr Arg Ile Gly Leu Gly Glu Ile His Leu Pro 740 745 750 Asp Glu Arg Lys Arg Pro Val Ser Asn Gly Arg Pro Ala Arg Pro Pro 755 760 765 Val Thr Ala Gly Leu Val Ala Ala Arg Ala Ala Gly Asp Pro Arg Pro 770 775 780 Pro His Asp Pro Thr His Pro Leu Ala Glu Ser Pro Arg Pro Ala Arg 785 790 795 800 Ser Ser Pro Ala Ser Ser Pro Glu Leu Thr Pro Ala Leu Glu Ala Thr 805 810 815 Ala Ala Pro Ala Ala Pro Ser Gly Ala Ser Thr Thr Arg Met Gln Ile 820 825 830 Gly Ser Ser Thr Glu Pro Pro Thr Thr Arg Leu Ala Ala Ala Gly Arg 835 840 845 Ser Val Gln Ser Pro Ala Ser Thr Pro Pro Pro Thr Pro Thr Pro Pro 850 855 860 Ser Ala Pro Ser Ala Gly Gln Thr Arg Ala Met Pro Leu Ala Ala Asn 865 870 875 880 Arg Ser Thr Asp Ala Ala Gly Asp Pro Ala Glu Pro Thr Ala Ala Leu 885 890 895 Pro Ile Ile Arg Ser Asp Gly Asp Asp Ser Glu Ala Ala Thr Glu Gln 900 905 910 Leu Asn Ala Arg Gly Thr Ser Asp Lys Thr Arg Gln Arg Arg Arg Gly 915 920 925 Gly Gly Ala Leu Ser Ala Gln Asp Leu Leu Arg Arg Glu Gly Arg Leu 930 935 940 26 2832 DNA Mycobacterium tuberculosis 26 gtgttcgcct ggtggggtcg aactgtgtac cgctaccggt tcatcgtaat cggggtcatg 60 gtcgctctat gcctcggcgg cggcgttttc gggctgagcc tcggcaagca cgtcacgcag 120 agcggcttct acgacgacgg cagccaatcg gtgcaagcat cggtgctggg cgaccaggtc 180 tacggccgag accgaagcgg tcacatcgtc gcgatcttcc aagccccagc cggcaagacc 240 gttgacgacc cggcctggtc aaagaaggtc gtcgacgagc tcaaccggtt ccagcaggat 300 caccccgacc aggtcttggg atgggccggc tacctgagag cgagtcaggc gaccggcatg 360 gccaccgccg acaagaagta caccttcgtt tccatcccgc tcaagggtga tgacgacgac 420 accatcctca acaactacaa ggccatcgca cccgacctgc agcggctcga cggaggcacg 480 gtgaagctcg ccgggctgca accggtggcc gaggcgttga ccggcaccat cgccaccgac 540 caacggcgaa tggaagtgct ggcgctgccg ttggtggcgg tggtgttgtt cttcgtgttc 600 ggcggcgtga tcgccgccgg cctaccggtg atggtcggag ggctgtgcat cgccggcgcg 660 ctgggcatca tgcggttcct cgcgatcttc ggtcccgtgc actatttcgc ccagcccgtg 720 gtgtcgctga tcggtctggg gatcgccatc gactacgggt tgttcatcgt gagccggttc 780 cgcgaagaga tcgccgaagg ctacgacacc gagacggcag tacggcgcac ggtgatcacc 840 gccggacgca cggtgacgtt ctcggcggtg ttgatcgtcg cgtcggcgat cggtctgctg 900 ctcttcccgc agggtttcct gaagtcgctg acctacgcca cgatcgcatc ggtgatgctg 960 tcggccatcc tgtctatcac cgtgttgccg gcctgtctgg ggatcctggg caaacacgtc 1020 gacgcgctcg gcgtgcggac cctgttccgg gtgcccttcc tggcgaactg gaagatttcg 1080 gccgcctacc tgaactggct cgccgaccgc ctgcagcgga ccaagacccg cgaagaggtc 1140 gaagccggct tctggggcaa gctggttaac cgggtgatga agcgcccagt gctgttcgcc 1200 gcaccgatcg tcatcatcat gattttgctg attatcccgg tgggcaagct gtcattgggc 1260 gggatcagcg agaagtactt gccgccgacc aattcggtgc gccaggcgca ggaggagttc 1320 gacaaactct tccccggata ccgcaccaat ccgctgacac tggtgatcca gaccagcaac 1380 catcaaccgg tcaccgacgc gcagatcgct gacatccgca gcaaggcgat ggcgatcggc 1440 ggattcatcg agccggacaa cgatccggcg aatatgtggc aagagcgtgc ctacgcggta 1500 ggcgcatcta aagatccatc ggtgcgcgtc ctgcagaacg ggttgatcaa cccggctgac 1560 gcgtcgaaga agctcaccga gctgcgcgcg atcaccccgc ccaaaggaat cacggtcttg 1620 gtcggtggaa ctcccgccct ggagctggat tcaatccacg gcctgttcgc gaagatgccg 1680 ctgatggtgg tcatcctgct gaccaccacg atcgtcttga tgttcttggc gttcggctcg 1740 gtggtgctgc caatcaaggc gacgctgatg agcgctctga cgctcgggtc caccatgggc 1800 atcctgacgt ggatattcgt cgacggacac ttttcgaagt ggctgaattt cacgccgacc 1860 ccgctgacag cgccggtgat cgggctgatc atcgcgctgg tcttcggcct atccaccgac 1920 tacgaggtgt tcttggtgtc ccggatggtc gaggcgcgag agcgcggcat gtcgacccag 1980 gaggcgatcc ggatcggcac cgcagccacc ggacgcatca ttaccgccgc ggcgctgatt 2040 gttgccgtcg tcgcgggcgc gttcgtgttc tccgacctgg tgatgatgaa gtatctggcc 2100 tttggactga tggcggcgct gctgctggac gcgaccgtgg tgcggatgtt tttagtgcca 2160 tcggtgatga agctgctcgg cgatgactgc tggtgggcac cgcgctgggc cagacgcctg 2220 cagacccgca tcgggctggg cgagatccac ctgcccgacg agcgcaagcg gcccgtcagc 2280 aacgggcgtc ccgcacgtcc tccggtcaca gctgggctgg ttgcggcgcg cgccgctggg 2340 gacccgcgcc caccgcacga tccgacccat ccgctggcgg agtcacctcg accggcccgc 2400 tcgagtccag caagctcacc ggagctcacg cctgccctgg aagcaactgc cgcgccggcg 2460 gcgccgtctg gggcgagcac cacacggatg cagatcgggt cgtcgacgga gccgccgaca 2520 acccgcctcg cggctgccgg tcggtccgtg cagtcgccag catccacgcc gccaccaacc 2580 ccgaccccgc catcggcccc gtctgccggt cagacccggg ctatgccgct tgcggcgaac 2640 cgctccacag acgcagccgg tgacccggcc gaacccaccg cggccctgcc aatcatacgg 2700 tcggacggcg acgactcaga ggcagccact gagcagctga atgcccgcgg cacgagcgat 2760 aagacgcgtc agcgccgccg cggcggcggc gccctgtccg cccaggatct gcttcgccgc 2820 gaaggacgcc tt 2832 27 920 PRT Mycobacterium tuberculosis 27 Met Pro Ser Pro Ala Gly Arg Leu His Arg Ile Arg Tyr Ile Arg Leu 1 5 10 15 Lys Lys Ser Ser Pro Asp Cys Arg Ala Thr Ile Thr Ser Gly Ser Ala 20 25 30 Asp Gly Gln Arg Arg Ser Pro Arg Leu Thr Asn Leu Leu Val Val Ala 35 40 45 Ala Trp Val Ala Ala Ala Val Ile Ala Asn Leu Leu Leu Thr Phe Thr 50 55 60 Gln Ala Glu Pro His Asp Thr Ser Pro Ala Leu Leu Pro Gln Asp Ala 65 70 75 80 Lys Thr Ala Ala Ala Thr Ser Arg Ile Ala Gln Ala Phe Pro Gly Thr 85 90 95 Gly Ser Asn Ala Ile Ala Tyr Leu Val Val Glu Gly Gly Ser Thr Leu 100 105 110 Glu Pro Gln Asp Gln Pro Tyr Tyr Asp Ala Ala Val Gly Ala Leu Arg 115 120 125 Ala Asp Thr Arg His Val Gly Ser Val Leu Asp Trp Trp Ser Asp Pro 130 135 140 Val Thr Ala Pro Leu Gly Thr Ser Pro Asp Gly Arg Ser Ala Thr Ala 145 150 155 160 Met Val Trp Leu Arg Gly Glu Ala Gly Thr Thr Gln Ala Ala Glu Ser 165 170 175 Leu Asp Ala Val Arg Ser Val Leu Arg Gln Leu Pro Pro Ser Glu Gly 180 185 190 Leu Arg Ala Ser Ile Val Val Pro Ala Ile Thr Asn Asp Met Pro Met 195 200 205 Gln Ile Thr Ala Trp Gln Ser Ala Thr Ile Val Thr Val Ala Ala Val 210 215 220 Ile Ala Val Leu Leu Leu Leu Arg Ala Arg Leu Ser Val Arg Ala Ala 225 230 235 240 Ala Ile Val Leu Leu Thr Ala Asp Leu Ser Leu Ala Val Ala Trp Pro 245 250 255 Leu Ala Ala Val Val Arg Gly His Asp Trp Gly Thr Asp Ser Val Phe 260 265 270 Ser Trp Thr Leu Ala Ala Val Leu Thr Ile Gly Thr Ile Thr Ala Ala 275 280 285 Thr Met Leu Ala Ala Arg Leu Gly Ser Asp Ala Gly His Ser Ala Ala 290 295 300 Pro Thr Tyr Arg Asp Ser Leu Pro Ala Phe Ala Leu Pro Gly Ala Cys 305 310 315 320 Val Ala Ile Phe Thr Gly Pro Leu Leu Leu Ala Arg Thr Pro Ala Leu 325 330 335 His Gly Val Gly Thr Ala Gly Leu Gly Val Phe Val Ala Leu Ala Ala 340 345 350 Ser Leu Thr Val Leu Pro Ala Leu Ile Ala Leu Ala Gly Ala Ser Arg 355 360 365 Gln Leu Pro Ala Pro Thr Thr Gly Ala Gly Trp Thr Gly Arg Leu Ser 370 375 380 Leu Pro Val Ser Ser Ala Ser Ala Leu Gly Thr Ala Ala Val Leu Ala 385 390 395 400 Ile Cys Met Leu Pro Ile Ile Gly Met Arg Trp Gly Val Ala Glu Asn 405 410 415 Pro Thr Arg Gln Gly Gly Ala Gln Val Leu Pro Gly Asn Ala Leu Pro 420 425 430 Asp Val Val Val Ile Lys Ser Ala Arg Asp Leu Arg Asp Pro Ala Ala 435 440 445 Leu Ile Ala Ile Asn Gln Val Ser His Arg Leu Val Glu Val Pro Gly 450 455 460 Val Arg Lys Val Glu Ser Ala Ala Trp Pro Ala Gly Val Pro Trp Thr 465 470 475 480 Asp Ala Ser Leu Ser Ser Ala Ala Gly Arg Leu Ala Asp Gln Leu Gly 485 490 495 Gln Gln Ala Gly Ser Phe Val Pro Ala Val Thr Ala Ile Lys Ser Met 500 505 510 Lys Ser Ile Ile Glu Gln Met Ser Gly Ala Val Asp Gln Leu Asp Ser 515 520 525 Thr Val Asn Val Thr Leu Ala Gly Ala Arg Gln Ala Gln Gln Tyr Leu 530 535 540 Asp Pro Met Leu Ala Ala Ala Arg Asn Leu Lys Asn Lys Thr Thr Glu 545 550 555 560 Leu Ser Glu Tyr Leu Glu Thr Ile His Thr Trp Ile Val Gly Phe Thr 565 570 575 Asn Cys Pro Asp Asp Val Leu Cys Thr Ala Met Arg Lys Val Ile Glu 580 585 590 Pro Tyr Asp Ile Val Val Thr Gly Met Asn Glu Leu Ser Thr Gly Ala 595 600 605 Asp Arg Ile Ser Ala Ile Ser Thr Gln Thr Met Ser Ala Leu Ser Ser 610 615 620 Ala Pro Arg Met Val Ala Gln Met Arg Ser Ala Leu Ala Gln Val Arg 625 630 635 640 Ser Phe Val Pro Lys Leu Glu Thr Thr Ile Gln Asp Ala Met Pro Gln 645 650 655 Ile Ala Gln Ala Ser Ala Met Leu Lys Asn Leu Ser Ala Asp Phe Ala 660 665 670 Asp Thr Gly Glu Gly Gly Phe His Leu Ser Arg Lys Asp Leu Ala Asp 675 680 685 Pro Ser Tyr Arg His Val Arg Glu Ser Met Phe Ser Ser Asp Gly Thr 690 695 700 Ala Thr Arg Leu Phe Leu Tyr Ser Asp Gly Gln Leu Asp Leu Ala Ala 705 710 715 720 Ala Ala Arg Ala Gln Gln Leu Glu Ile Ala Ala Gly Lys Ala Met Lys 725 730 735 Tyr Gly Ser Leu Val Asp Ser Gln Val Thr Val Gly Gly Ala Ala Gln 740 745 750 Ile Ala Ala Ala Val Arg Asp Ala Leu Ile His Asp Ala Val Leu Leu 755 760 765 Ala Val Ile Leu Leu Thr Val Val Ala Leu Ala Ser Met Trp Arg Gly 770 775 780 Ala Val His Gly Ala Ala Val Gly Val Gly Val Leu Ala Ser Tyr Leu 785 790 795 800 Ala Ala Leu Gly Val Ser Ile Ala Leu Trp Gln His Leu Leu Asp Arg 805 810 815 Glu Leu Asn Ala Leu Val Pro Leu Val Ser Phe Ala Val Leu Ala Ser 820 825 830 Cys Gly Val Pro Tyr Leu Val Ala Gly Ile Lys Ala Gly Arg Ile Ala 835 840 845 Asp Glu Ala Thr Gly Ala Arg Ser Lys Gly Ala Val Ser Gly Arg Gly 850 855 860 Ala Val Ala Pro Leu Ala Ala Leu Gly Gly Val Phe Gly Ala Gly Leu 865 870 875 880 Val Leu Val Ser Gly Gly Ser Phe Ser Val Leu Ser Gln Ile Gly Thr 885 890 895 Val Val Val Leu Gly Leu Gly Val Leu Ile Thr Val Gln Arg Ala Trp 900 905 910 Leu Pro Thr Thr Pro Gly Arg Arg 915 920 28 2760 DNA Mycobacterium tuberculosis 28 atgcctagtc cggctggccg tctacacaga attcggtata tccgtttgaa aaagtcctcc 60 ccggactgcc gcgccaccat caccagcggg tcagccgacg gtcagcgaag gtcaccccgg 120 ctcaccaacc tgctcgtcgt cgccgcctgg gttgccgcgg cggtgatcgc aaatctgctt 180 ctcacgttca cgcaagcaga accgcacgac accagcccgg cgctgctgcc acaagatgcc 240 aagacagccg ccgccaccag ccggattgcg caggctttcc ccggcaccgg tagcaacgct 300 atcgcctatc tcgtcgtgga aggcggcagc acgcttgagc cgcaggacca gccttactac 360 gacgccgccg tcggtgccct gcgcgccgac acccgccacg tgggatccgt cctcgactgg 420 tggtcagatc ccgtcaccgc cccgctggga accagccccg acggccgctc cgctacggcc 480 atggtgtggc tgcggggcga ggcgggcacc acccaagctg ccgaatccct cgatgccgtc 540 cgatcggtgc tgcgccagtt accgcccagt gaggggcttc gcgccagcat cgtggtcccg 600 gcaatcacca acgacatgcc gatgcagata accgcctggc agagcgcgac gatcgtgacc 660 gttgcggcgg tgatcgccgt cctactgctg ctgcgggcgc gcctgtcggt gcgggccgcg 720 gcgatcgtgc tgctgaccgc ggacttgtcg cttgcggtgg cctggccgct ggccgcggtg 780 gtgcggggac acgattgggg aaccgattcg gtattttctt ggacgctggc cgcggtcctg 840 acgatcggaa ccatcaccgc agccaccatg ctggccgcgc ggctcgggtc cgacgcaggt 900 cattcggccg cgcccacata ccgcgacagc ctgcccgcgt tcgccctgcc cggggcgtgt 960 gtcgccatat tcaccggccc gctgctgctg gcccgaaccc cagcgctgca cggagttggc 1020 actgccgggc taggtgtctt tgtggcactt gcggcttcgt tgacggtgct gcctgccctg 1080 atcgcgcttg ccggagcgtc acggcagtta ccggcaccaa ccacgggtgc cggctggaca 1140 ggccggttgt cgctacccgt ctcttctgct tcggccctgg gcacagcggc agtgctggcg 1200 atctgcatgc tacccatcat cgggatgcgg tggggtgtgg ccgagaaccc gacaaggcaa 1260 ggcggcgcac aagtccttcc ggggaatgcg cttcccgatg tggtggtgat caaatccgct 1320 cgggacctga gggacccagc cgcgctcatc gccatcaacc aggtcagcca ccgtctggtg 1380 gaggttcccg gtgtgcgcaa ggtggagtcg gcggcatggc cggccggtgt cccgtggacc 1440 gacgcctcgc tcagttccgc ggccggcagg ctcgccgacc agctgggtca gcaggccgga 1500 tcgttcgtgc cggcggtgac tgcgatcaaa tcgatgaagt ccataatcga acagatgagc 1560 ggcgcggtcg accaactgga cagcaccgtg aacgtgactc tcgccggggc aaggcaagca 1620 cagcaatacc tcgatcccat gctcgccgcc gcgcggaacc tcaaaaacaa aaccaccgaa 1680 ctgtcggaat acctggaaac gatccacacc tggattgtcg gcttcacaaa ctgccccgac 1740 gacgtcctgt gcacggccat gcgcaaggtc attgaaccct acgacatcgt ggtcaccggc 1800 atgaacgagc tgtccactgg cgccgaccgc atctccgcga tatcgacaca gacaatgagc 1860 gcgttgtcct cggcaccgcg gatggtggcg cagatgcggt cggcgctagc acaggtgcgc 1920 tcgttcgtac ccaagctgga aacaaccatc caggacgcca tgccgcaaat agcgcaggcg 1980 tcggcgatgc tgaagaatct cagcgccgat ttcgccgata ccggtgaggg cggcttccac 2040 ctgtccagga aggacctggc ggacccgtcg taccggcacg tacgggaatc gatgttctcg 2100 tcagacggaa ccgccacccg gctgttcctc tattctgacg gacaactgga ccttgctgcg 2160 gcagcacgcg cgcagcagct cgagatcgcc gcgggcaagg cgatgaaata cggaagcctg 2220 gtcgacagcc aggtcacggt gggtggggcc gcgcaaatag ccgcggctgt ccgcgatgcc 2280 ctcatccacg atgctgtgct actggccgtt atcttgctca cggtagtggc tctggccagc 2340 atgtggcgcg gtgccgtcca cggtgctgcg gttggcgtgg gtgtgctggc ctcttacctc 2400 gccgccctgg gggtctcgat tgcactgtgg caacacctac tggatcgcga gctcaacgcc 2460 ttggtcccgc tggtgtcgtt cgccgtcctc gcttcgtgcg gcgtcccgta tctcgttgcc 2520 ggcatcaaag ccggtcgtat cgccgacgag gcaacgggtg cgcggtccaa gggggcggta 2580 tccgggcggg gagcggttgc gccgcttgcg gcgctcggtg gcgtattcgg cgctggcctg 2640 gtgctggtgt cgggaggttc cttcagcgtg ctcagtcaga ttggcacggt tgttgtgctc 2700 ggtctgggcg tgctgatcac ggtgcagcga gcgtggcttc cgaccacgcc agggcggcgt 2760 29 84 PRT Mycobacterium tuberculosis 29 Met Ala Lys Ser Ser Lys Arg Arg Pro Ala Pro Glu Lys Pro Val Lys 1 5 10 15 Thr Arg Lys Cys Val Phe Cys Ala Lys Lys Asp Gln Ala Ile Asp Tyr 20 25 30 Lys Asp Thr Ala Leu Leu Arg Thr Tyr Ile Ser Glu Arg Gly Lys Ile 35 40 45 Arg Ala Arg Arg Val Thr Gly Asn Cys Val Gln His Gln Arg Asp Ile 50 55 60 Ala Leu Ala Val Lys Asn Ala Arg Glu Val Ala Leu Leu Pro Phe Thr 65 70 75 80 Ser Ser Val Arg 30 252 DNA Mycobacterium tuberculosis 30 atggccaagt ccagcaagcg gcgcccggct ccggaaaagc cggtcaagac gcgtaaatgc 60 gtgttctgcg cgaagaagga ccaagcgatc gactacaagg acaccgcgct gttgcgcacc 120 tacatcagcg agcgcggcaa gatccgcgcg cgtcgggtca cgggcaactg cgtgcagcac 180 cagcgagaca tcgcgctcgc ggtgaagaac gcccgcgagg tggcgctgct gccctttacg 240 tcttcggtgc gg 252 31 280 PRT Mycobacterium tuberculosis 31 Val Pro His Ser Trp Thr Pro Thr Ser Val Met Thr Pro Pro Leu Val 1 5 10 15 Val Ala Ala Phe Arg Pro Val Gly His Tyr Arg Leu Ala Thr Asp Arg 20 25 30 Ala Gly Gly Pro Cys Ser Pro Pro Ala Thr Gly Ala Lys Leu Thr Ser 35 40 45 Ser Val Ala Ser Arg Pro Thr Val Gly Thr Lys Pro Gln Trp Trp His 50 55 60 Thr Leu Val Met Ser Met Ser Leu Thr Ala Gly Arg Gly Pro Gly Arg 65 70 75 80 Pro Pro Ala Ala Lys Ala Asp Glu Thr Arg Lys Arg Ile Leu His Ala 85 90 95 Ala Arg Gln Val Phe Ser Glu Arg Gly Tyr Asp Gly Ala Thr Phe Gln 100 105 110 Glu Ile Ala Val Arg Ala Asp Leu Thr Arg Pro Ala Ile Asn His Tyr 115 120 125 Phe Ala Asn Lys Arg Val Leu Tyr Gln Glu Val Val Glu Gln Thr His 130 135 140 Glu Leu Val Ile Val Ala Gly Ile Glu Arg Ala Arg Arg Glu Pro Thr 145 150 155 160 Leu Met Gly Arg Leu Ala Val Val Val Asp Phe Ala Met Glu Ala Asp 165 170 175 Ala Gln Tyr Pro Ala Ser Thr Ala Phe Leu Ala Thr Thr Val Leu Glu 180 185 190 Ser Gln Arg His Pro Glu Leu Ser Arg Thr Glu Asn Asp Ala Val Arg 195 200 205 Ala Thr Arg Glu Phe Leu Val Trp Ala Val Asn Asp Ala Ile Glu Arg 210 215 220 Gly Glu Leu Ala Ala Asp Val Asp Val Ser Ser Leu Ala Glu Thr Leu 225 230 235 240 Leu Val Val Leu Cys Gly Val Gly Phe Tyr Ile Gly Phe Val Gly Ser 245 250 255 Tyr Gln Arg Met Ala Thr Ile Thr Asp Ser Phe Gln Gln Leu Leu Ala 260 265 270 Gly Thr Leu Trp Arg Pro Pro Thr 275 280 32 840 DNA Mycobacterium tuberculosis 32 gtgccgcact cttggacccc gacctctgtc atgacgccgc cgctcgtcgt ggccgcgttc 60 aggccggtcg gccattaccg actcgcaacg gacagagccg gtgggccctg ctcgcccccg 120 gcgaccggag ccaagctgac aagttccgta gcatcccgcc caacggtagg taccaagccg 180 cagtggtggc acactttagt gatgtcaatg tcgctcacgg ccggtcgcgg cccgggacgt 240 cccccggcgg cgaaagcaga tgagactcgg aagcgtattc tgcacgccgc ccgtcaagtg 300 ttcagcgaac gtggttatga cggcgcgact tttcaggaga tcgccgtccg cgccgacctg 360 acccgaccgg cgatcaacca ctacttcgcc aacaagcggg tgctctacca agaggtggtg 420 gagcaaaccc acgaactcgt cattgtggcc ggcatcgaac gggcacgccg cgagccgacc 480 ttgatggggc ggctggcggt cgtcgttgac ttcgcgatgg aggccgatgc ccagtatccc 540 gcctcgaccg cgttcctggc caccaccgtg ctcgaatccc agcggcatcc agaattgagt 600 cggaccgaaa acgatgcggt gcgagcaacc cgagaattcc tggtttgggc tgtcaatgat 660 gcgatcgaac gcggtgaact agccgccgac gtcgatgtct cttcgttggc cgagacgctg 720 ttggtcgtgt tgtgtggcgt gggcttctat atcggttttg tcgggagcta tcagcggatg 780 gcgaccatca ccgattcgtt ccagcagctg ttggccggca cgctctggcg gcctccgacc 840 33 663 PRT Mycobacterium tuberculosis 33 Val Thr Leu Ala Ile Pro Ser Gly Ile Asp Leu Ser His Ile Asp Ala 1 5 10 15 Asp Ala Arg Pro Gln Asp Asp Leu Phe Gly His Val Asn Gly Arg Trp 20 25 30 Leu Ala Glu His Glu Ile Pro Ala Asp Arg Ala Thr Asp Gly Ala Phe 35 40 45 Arg Ser Leu Phe Asp Arg Ala Glu Thr Gln Val Arg Asp Leu Ile Ile 50 55 60 Gln Ala Ser Gln Ala Gly Ala Ala Val Gly Thr Asp Ala Gln Arg Ile 65 70 75 80 Gly Asp Leu Tyr Ala Ser Phe Leu Asp Glu Glu Ala Val Glu Arg Ala 85 90 95 Gly Val Gln Pro Leu His Asp Glu Leu Ala Thr Ile Asp Ser Ala Ala 100 105 110 Asp Ala Thr Glu Leu Ala Ala Ala Leu Gly Thr Leu Gln Arg Ala Gly 115 120 125 Val Gly Gly Gly Ile Gly Val Tyr Val Asp Thr Asp Ser Lys Asp Ser 130 135 140 Thr Arg Tyr Leu Val His Phe Thr Gln Ser Gly Ile Gly Leu Pro Asp 145 150 155 160 Glu Ser Tyr Tyr Arg Asp Glu Gln His Ala Ala Val Leu Ala Ala Tyr 165 170 175 Pro Gly His Ile Ala Arg Met Phe Gly Leu Val Tyr Gly Gly Glu Ser 180 185 190 Arg Asp His Ala Lys Thr Ala Asp Arg Ile Val Ala Leu Glu Thr Lys 195 200 205 Leu Ala Asp Ala His Trp Asp Val Val Lys Arg Arg Asp Ala Asp Leu 210 215 220 Gly Tyr Asn Leu Arg Thr Phe Ala Gln Leu Gln Thr Glu Gly Ala Gly 225 230 235 240 Phe Asp Trp Val Ser Trp Val Thr Ala Leu Gly Ser Ala Pro Asp Ala 245 250 255 Met Thr Glu Leu Val Val Arg Gln Pro Asp Tyr Leu Val Thr Phe Ala 260 265 270 Ser Leu Trp Ala Ser Val Asn Val Glu Asp Trp Lys Cys Trp Ala Arg 275 280 285 Trp Arg Leu Ile Arg Ala Arg Ala Pro Trp Leu Thr Arg Ala Leu Val 290 295 300 Ala Glu Asp Phe Glu Phe Tyr Gly Arg Thr Leu Thr Gly Ala Gln Gln 305 310 315 320 Leu Arg Asp Arg Trp Lys Arg Gly Val Ser Leu Val Glu Asn Leu Met 325 330 335 Gly Asp Ala Val Gly Lys Leu Tyr Val Gln Arg His Phe Pro Pro Asp 340 345 350 Ala Lys Ser Arg Ile Asp Thr Leu Val Asp Asn Leu Gln Glu Ala Tyr 355 360 365 Arg Ile Ser Ile Ser Glu Leu Asp Trp Met Thr Pro Gln Thr Arg Gln 370 375 380 Arg Ala Leu Ala Lys Leu Asn Lys Phe Thr Ala Lys Val Gly Tyr Pro 385 390 395 400 Ile Lys Trp Arg Asp Tyr Ser Lys Leu Ala Ile Asp Arg Asp Asp Leu 405 410 415 Tyr Gly Asn Val Gln Arg Gly Tyr Ala Val Asn His Asp Arg Glu Leu 420 425 430 Ala Lys Leu Phe Gly Pro Val Asp Arg Asp Glu Trp Phe Met Thr Pro 435 440 445 Gln Thr Val Asn Ala Tyr Tyr Asn Pro Gly Met Asn Glu Ile Val Phe 450 455 460 Pro Ala Ala Ile Leu Gln Pro Pro Phe Phe Asp Pro Gln Ala Asp Glu 465 470 475 480 Ala Ala Asn Tyr Gly Gly Ile Gly Ala Val Ile Gly His Glu Ile Gly 485 490 495 His Gly Phe Asp Asp Gln Gly Ala Lys Tyr Asp Gly Asp Gly Asn Leu 500 505 510 Val Asp Trp Trp Thr Asp Asp Asp Arg Thr Glu Phe Ala Ala Arg Thr 515 520 525 Lys Ala Leu Ile Glu Gln Tyr His Ala Tyr Thr Pro Arg Asp Leu Val 530 535 540 Asp His Pro Gly Pro Pro His Val Gln Gly Ala Phe Thr Ile Gly Glu 545 550 555 560 Asn Ile Gly Asp Leu Gly Gly Leu Ser Ile Ala Leu Leu Ala Tyr Gln 565 570 575 Leu Ser Leu Asn Gly Asn Pro Ala Pro Val Ile Asp Gly Leu Thr Gly 580 585 590 Met Gln Arg Val Phe Phe Gly Trp Ala Gln Ile Trp Arg Thr Lys Ser 595 600 605 Arg Ala Ala Glu Ala Ile Arg Arg Leu Ala Val Asp Pro His Ser Pro 610 615 620 Pro Glu Phe Arg Cys Asn Gly Val Val Arg Asn Val Asp Ala Phe Tyr 625 630 635 640 Gln Ala Phe Asp Val Thr Glu Asp Asp Ala Leu Phe Leu Asp Pro Gln 645 650 655 Arg Arg Val Arg Ile Trp Asn 660 34 1989 DNA Mycobacterium tuberculosis 34 gtgacacttg ccatcccctc gggtatcgac ctgagccaca tcgacgctga tgcccgaccc 60 caagacgacc tgttcggcca cgttaacggc cgctggctgg ctgaacacga gataccagcg 120 gaccgagcga ccgacggcgc cttccgtagc ctgttcgacc gcgccgagac acaagtgcga 180 gacctgatca tccaggccag ccaagcaggt gctgcggtag gcaccgatgc gcagcgcatc 240 ggcgacctct acgccagctt cctcgacgag gaagccgtcg agcgcgcagg ggtgcaaccg 300 ctgcacgacg aattggccac gattgacagc gcggccgacg ccaccgaatt ggccgccgcc 360 cttggcactc tgcaacgtgc cggcgtgggc ggcggcatcg gagtctatgt cgataccgat 420 tccaaagact cgacccgtta cttggtgcat ttcacccaat ccggcatcgg attacccgac 480 gagtcctact accgtgacga gcaacacgcc gccgtgctag cggcctaccc ggggcacatc 540 gcccggatgt tcggcctggt gtacgggggc gagagccgtg accatgccaa aaccgcggac 600 cgcatcgtcg cgctggagac caaactcgcc gacgcgcatt gggatgtggt gaagcgccgc 660 gacgccgacc ttggctacaa cctgcgcacg tttgcccagc tgcagaccga aggggcgggt 720 ttcgactggg tcagctgggt gaccgcattg gggagcgctc cggacgccat gacggaactg 780 gttgtgcgcc aacctgatta cctcgtcacc tttgcctcgc tgtgggcgag cgttaacgtt 840 gaagactgga aatgctgggc gcgttggcgt ttgatccgcg cccgggcccc ctggctgacc 900 cgcgccctgg tcgccgagga cttcgaattc tacggccgca cgcttaccgg cgcacagcag 960 cttcgggacc gttggaagcg tggggtgtca ctggtggaga acctgatggg cgatgccgtc 1020 ggaaagctct atgtacaacg ccatttcccg ccggatgcca agtcccgcat cgacaccctg 1080 gtggacaacc tgcaggaggc gtatcggatc agcatcagcg agctggattg gatgacgccg 1140 cagacccggc aacgcgcgct agcgaagctg aacaagttca ccgccaaagt cggctatccg 1200 atcaagtggc gcgactactc gaagctggcg atcgaccgcg acgacctcta cggtaacgtc 1260 cagcgcggct acgccgtcaa ccatgaccgc gagctagcca agcttttcgg cccggtcgac 1320 cgcgacgagt ggttcatgac accacaaacc gtcaacgcct actacaaccc ggggatgaac 1380 gaaatcgtct tccccgcagc gattttacag ccaccatttt tcgatccgca ggccgacgag 1440 gccgccaact acggcgggat cggggcggtg atcgggcacg agatcgggca cggtttcgac 1500 gatcagggcg ccaaatacga cggcgacggc aatctggtcg attggtggac cgacgacgat 1560 cgcaccgagt tcgccgcccg caccaaagcg ttgatcgagc agtaccacgc ttacacgccg 1620 cgcgatctcg tcgaccaccc cggcccgcct catgtgcaag gcgcgttcac cataggcgag 1680 aacatcggcg acctgggcgg gctgtcgatc gccctgctgg cttaccagct ctcgctgaac 1740 ggcaaccccg ctccggttat cgacgggctg accggcatgc aacgggtgtt cttcggctgg 1800 gcacaaatat ggcgaaccaa atcgcgtgca gccgaagcaa tccgccggtt ggcggtcgat 1860 ccgcactccc cgccggagtt ccggtgcaac ggtgtggttc gcaacgtgga cgctttttat 1920 caggccttcg acgtcaccga ggatgacgcg ctgtttctgg acccgcagcg cagggtccgg 1980 atctggaac 1989 35 275 PRT Mycobacterium tuberculosis 35 Met Ser Asn Ala Pro Glu Pro Asp Arg Ser Ala Gly Glu Ser Gly Ser 1 5 10 15 Glu Pro Ala Gly Glu Arg Ser Ala Asp Pro Gly Glu Glu Arg Thr Glu 20 25 30 Ser Tyr Pro Leu Val Pro His Asp Ala Glu Thr Glu Thr Val Val Ile 35 40 45 Thr Thr Ser Asp Asn Asp Ala Ala Val Thr Gln Pro Glu Ala Gln Arg 50 55 60 Glu Arg Arg Phe Thr Ala Pro Gly Phe Asp Ala Lys Glu Thr Gln Val 65 70 75 80 Ile Val Thr Ala His Glu Ala Ala Thr Glu Val Phe Gln Thr Asn Gln 85 90 95 Ala Pro Thr Thr Pro Pro Arg Met Pro Thr Gly Met Pro Pro Lys Thr 100 105 110 Ala Val Pro Gln Ser Ile Pro Pro Arg Thr Glu Ala Thr Ser Val Arg 115 120 125 Gln Arg Thr Trp Gly Trp Ala Leu Ala Val Val Val Ile Val Leu Ala 130 135 140 Leu Ala Ala Ile Ala Ile Leu Gly Thr Val Leu Leu Thr Arg Gly Lys 145 150 155 160 His Ser Lys Met Ser Gln Glu Asp Gln Val Arg Gln Ala Ile Gln Ser 165 170 175 Leu Asp Ile Ala Ile Gln Thr Gly Asp Leu Thr Ala Leu Arg Ser Leu 180 185 190 Thr Cys Gly Ser Thr Arg Asp Gly Tyr Val Asp Tyr Asp Glu Arg Asp 195 200 205 Trp Ala Glu Thr Tyr Arg Arg Val Ser Ala Ala Lys Gln Tyr Pro Val 210 215 220 Ile Ala Ser Ile Asp Gln Val Val Val Asn Gly Ala His Ala Glu Ala 225 230 235 240 Asn Val Thr Thr Phe Met Ala Phe Asp Pro Gln Val Arg Ser Thr Arg 245 250 255 Ser Leu Asp Leu Gln Phe Arg Asp Asp Gln Trp Lys Ile Cys Gln Ser 260 265 270 Ser Ser Asn 275 36 825 DNA Mycobacterium tuberculosis 36 atgtccaacg cacccgagcc agaccgctca gccggtgaat ccgggagcga accggccggc 60 gagcggtccg ccgatcctgg cgaggaacgc accgaaagct accccctggt gcctcacgac 120 gccgaaaccg agaccgtggt gatcaccacc tccgacaacg atgccgcggt tacgcaaccg 180 gaagcgcagc gcgaacgccg tttcaccgcg cccggcttcg acgccaagga gacccaggtg 240 atcgtcacgg cccacgaggc agccaccgag gttttccaaa ccaaccaggc gccgaccacc 300 ccgccgcgga tgccaaccgg aatgcccccg aaaactgctg tgccacaatc aatcccgcca 360 cggacggagg cgacgtcagt ccggcaacgc acctggggct gggcgctggc ggtggtagtg 420 atcgtgctgg cgttggcggc aatcgcgatc ctgggcaccg tgctgctgac ccgcggcaaa 480 cattcgaaga tgtcgcagga agatcaggtg cggcaggcca tccagagctt ggacatcgcc 540 atccagaccg gcgacctgac cgcgctgcgt tccctgactt gtggctccac ccgcgatggc 600 tacgtggatt atgacgagcg tgattgggcc gaaacctatc gccgggtttc ggcggccaaa 660 caatatccgg tcatcgccag catcgaccag gtcgtcgtca acggcgcgca cgccgaggcc 720 aatgtcacca ctttcatggc gttcgatccc caggtccgct cgacccgcag cctcgaccta 780 cagtttcgcg acgatcagtg gaagatctgc cagtcctcca gcaac 825 37 158 PRT Mycobacterium tuberculosis 37 Val Ala Leu Ser Ala Asp Ile Val Gly Met His Tyr Arg Tyr Pro Asp 1 5 10 15 His Tyr Glu Val Glu Arg Glu Lys Ile Arg Glu Tyr Ala Val Ala Val 20 25 30 Gln Asn Asp Asp Ala Trp Tyr Phe Glu Glu Asp Gly Ala Ala Glu Leu 35 40 45 Gly Tyr Lys Gly Leu Leu Ala Pro Leu Thr Phe Ile Cys Val Phe Gly 50 55 60 Tyr Lys Ala Gln Ala Ala Phe Phe Lys His Ala Asn Ile Ala Thr Ala 65 70 75 80 Glu Ala Gln Ile Val Gln Val Asp Gln Val Leu Lys Phe Glu Lys Pro 85 90 95 Ile Val Ala Gly Asp Lys Leu Tyr Cys Asp Val Tyr Val Asp Ser Val 100 105 110 Arg Glu Ala His Gly Thr Gln Ile Ile Val Thr Lys Asn Ile Val Thr 115 120 125 Asn Glu Glu Gly Asp Leu Val Gln Glu Thr Tyr Thr Thr Leu Ala Gly 130 135 140 Arg Ala Gly Glu Asp Gly Glu Gly Phe Ser Asp Gly Ala Ala 145 150 155 38 474 DNA Mycobacterium tuberculosis 38 gtggcgttga gcgcagacat cgttgggatg cattaccggt atcccgacca ctacgaggtg 60 gagcgggaga agattcgcga gtacgccgtc gccgttcaaa acgacgacgc gtggtatttc 120 gaggaggacg gcgccgccga actcgggtat aagggcttgc tggctccgtt gacgtttatc 180 tgtgtgttcg gctacaaggc ccaggcggcg ttcttcaagc atgcgaacat cgcgaccgcg 240 gaggcgcaga tcgtccaggt agaccaagtg ctgaaattcg agaaaccgat cgtggcgggc 300 gacaagctgt actgcgacgt ctatgtggat tcggtgcgtg aggcgcacgg cacccagatc 360 atcgtgacca agaacatcgt caccaacgag gaaggtgacc tcgtgcagga gacctatacg 420 accctggcgg gccgtgccgg cgaggatgga gagggatttt ctgatggcgc tgcg 474 39 318 PRT Mycobacterium tuberculosis 39 Val His Arg Leu Arg Ala Ala Glu His Pro Arg Pro Asp Tyr Val Leu 1 5 10 15 Leu His Ile Ser Asp Thr His Leu Ile Gly Gly Asp Arg Arg Leu Tyr 20 25 30 Gly Ala Val Asp Ala Asp Asp Arg Leu Gly Glu Leu Leu Glu Gln Leu 35 40 45 Asn Gln Ser Gly Leu Arg Pro Asp Ala Ile Val Phe Thr Gly Asp Leu 50 55 60 Ala Asp Lys Gly Glu Pro Ala Ala Tyr Arg Lys Leu Arg Gly Leu Val 65 70 75 80 Glu Pro Phe Ala Ala Gln Leu Gly Ala Glu Leu Val Trp Val Met Gly 85 90 95 Asn His Asp Asp Arg Ala Glu Leu Arg Lys Phe Leu Leu Asp Glu Ala 100 105 110 Pro Ser Met Ala Pro Leu Asp Arg Val Cys Met Ile Asp Gly Leu Arg 115 120 125 Ile Ile Val Leu Asp Thr Ser Val Pro Gly His His His Gly Glu Ile 130 135 140 Arg Ala Ser Gln Leu Gly Trp Leu Ala Glu Glu Leu Ala Thr Pro Ala 145 150 155 160 Pro Asp Gly Thr Ile Leu Ala Leu His His Pro Pro Ile Pro Ser Val 165 170 175 Leu Asp Met Ala Val Thr Val Glu Leu Arg Asp Gln Ala Ala Leu Gly 180 185 190 Arg Val Leu Arg Gly Thr Asp Val Arg Ala Ile Leu Ala Gly His Leu 195 200 205 His Tyr Ser Thr Asn Ala Thr Phe Val Gly Ile Pro Val Ser Val Ala 210 215 220 Ser Ala Thr Cys Tyr Thr Gln Asp Leu Thr Val Ala Ala Gly Gly Thr 225 230 235 240 Arg Gly Arg Asp Gly Ala Gln Gly Cys Asn Leu Val His Val Tyr Pro 245 250 255 Asp Thr Val Val His Ser Val Ile Pro Leu Gly Gly Gly Glu Thr Val 260 265 270 Gly Thr Phe Val Ser Pro Gly Gln Ala Arg Arg Lys Ile Ala Glu Ser 275 280 285 Gly Ile Phe Ile Glu Pro Ser Arg Arg Asp Ser Leu Phe Lys His Pro 290 295 300 Pro Met Val Leu Thr Ser Ser Ala Pro Arg Ser Pro Val Asp 305 310 315 40 954 DNA Mycobacterium tuberculosis 40 gtgcatagac ttagggccgc ggagcatccg cggccggatt acgttctctt acatatcagc 60 gacactcatc tcatcggggg ggatcgtcgg ctctacgggg cggtggacgc cgacgaccgg 120 ctgggcgaac tgctcgaaca gttgaaccaa tccggccttc gtcccgatgc gatcgtcttc 180 accggcgatt tggccgataa gggcgaaccg gcggcatacc gcaagctccg aggcctggtc 240 gagccgttcg cggcgcagtt gggcgccgag ctcgtctggg tgatgggtaa ccacgacgac 300 cgggccgaac tacgcaaatt cttgctggac gaagcgccat cgatggcgcc gctagaccgg 360 gtgtgcatga tcgacggtct gcgcatcatc gtgttggata cctcggtacc cggacatcat 420 cacggcgaaa tccgcgcgtc ccaattgggt tggcttgctg aagagttggc cacgccagcg 480 ccggacggca ccattttggc gttgcatcat ccgccgattc cgagtgtttt ggatatggcc 540 gtcacggtgg agctgcgcga ccaggctgcg cttgggcgag tgctgcgggg cactgacgtt 600 cgcgccattt tggccgggca cctgcactac tcgacgaatg ccaccttcgt cgggatccca 660 gtgtcggttg cctcggcgac ttgctacacc caggacctga ccgtcgctgc tggaggaacg 720 cgtggcagag acggcgccca aggttgcaac ctggtgcacg tctatccgga caccgtcgtg 780 cattcggtga ttccgctggg cggcggagaa acggtcggca cctttgtctc acccgggcag 840 gcgcgacgca aaatcgccga gagcggcatt ttcatcgaac cgtcgcgtcg cgattcgcta 900 ttcaagcacc ctccgatggt gctgacgtcc tcggcaccgc gaagtcccgt cgac 954 41 129 PRT Mycobacterium tuberculosis 41 Leu Asn Ala Ala Met Asn Leu Lys Arg Glu Phe Val His Arg Val Gln 1 5 10 15 Arg Phe Val Val Asn Pro Ile Gly Arg Gln Leu Pro Met Thr Met Leu 20 25 30 Glu Thr Ile Gly Arg Lys Thr Gly Gln Pro Arg Arg Thr Ala Val Gly 35 40 45 Gly Arg Val Val Asp Asn Gln Phe Trp Met Val Ser Glu His Gly Glu 50 55 60 His Ser Asp Tyr Val Tyr Asn Ile Lys Ala Asn Pro Ala Val Arg Val 65 70 75 80 Arg Ile Gly Gly Arg Trp Arg Ser Gly Thr Ala Tyr Leu Leu Pro Asp 85 90 95 Asp Asp Pro Arg Gln Arg Leu Arg Gly Leu Pro Arg Leu Asn Ser Ala 100 105 110 Gly Val Arg Ala Met Gly Thr Asp Leu Leu Thr Ile Arg Val Asp Leu 115 120 125 Asp 42 387 DNA Mycobacterium tuberculosis 42 ttgaatgcag ctatgaatct caagcgggaa ttcgtccatc gcgtgcaacg gttcgtggtc 60 aatccaatcg gccggcaact gccgatgacc atgctcgaaa ccatcggccg caaaacggga 120 cagccgcggc gtaccgcggt gggcgggcgc gtcgtagaca accagttctg gatggtgtcc 180 gagcacggcg agcattccga ttacgtctac aacatcaagg ccaaccccgc cgtgcgggtc 240 cgcatcggcg gccgatggcg cagtgggacc gcctacctgc tacccgacga cgatccgagg 300 cagcggctgc gcggcctacc ccggctgaac agtgccggcg tacgcgcgat gggcaccgac 360 ttgctgacca tccgggtgga tttggac 387 43 27 PRT Mycobacterium tuberculosis 43 Met Pro Ala Ser Ser Leu Gly Thr Gly Ser Pro Ala Ala Asp Arg Leu 1 5 10 15 Asp Ala Thr His Glu Arg Arg Arg Glu Val Ile 20 25 44 81 DNA Mycobacterium tuberculosis 44 atgccggcat cgagtctggg taccgggtcg cccgccgccg acaggctcga cgccacccac 60 gagcgtcggc gtgaggtcat t 81 45 287 PRT Mycobacterium tuberculosis 45 Val Thr Val Ser Asp Ser Pro Ala Gln Arg Gln Thr Pro Pro Gln Thr 1 5 10 15 Pro Gly Gly Thr Ala Pro Arg Ala Arg Thr Ala Ala Phe Phe Asp Leu 20 25 30 Asp Lys Thr Ile Ile Ala Lys Ser Ser Thr Leu Ala Phe Ser Lys Pro 35 40 45 Phe Phe Ala Gln Gly Leu Leu Asn Arg Arg Ala Val Leu Lys Ser Ser 50 55 60 Tyr Ala Gln Phe Ile Phe Leu Leu Ser Gly Ala Asp His Asp Gln Met 65 70 75 80 Asp Arg Met Arg Thr His Leu Thr Asn Met Cys Ala Gly Trp Asp Val 85 90 95 Ala Gln Val Arg Ser Ile Val Asn Glu Thr Leu His Asp Ile Val Thr 100 105 110 Pro Leu Val Phe Ala Glu Ala Ala Asp Leu Ile Ala Ala His Lys Leu 115 120 125 Cys Gly Arg Asp Val Val Val Val Ser Ala Ser Gly Glu Glu Ile Val 130 135 140 Gly Pro Ile Ala Arg Ala Leu Gly Ala Thr His Ala Met Ala Thr Arg 145 150 155 160 Met Ile Val Glu Asp Gly Lys Tyr Thr Gly Glu Val Ala Phe Tyr Cys 165 170 175 Tyr Gly Glu Gly Lys Ala Gln Ala Ile Arg Glu Leu Ala Ala Ser Glu 180 185 190 Gly Tyr Pro Leu Glu His Cys Tyr Ala Tyr Ser Asp Ser Ile Thr Asp 195 200 205 Leu Pro Met Leu Glu Ala Val Gly His Ala Ser Val Val Asn Pro Asp 210 215 220 Arg Gly Leu Arg Lys Glu Ala Ser Val Arg Gly Trp Pro Val Leu Ser 225 230 235 240 Phe Ser Arg Pro Val Ser Leu Arg Asp Arg Ile Pro Ala Pro Ser Ala 245 250 255 Ala Ala Ile Ala Thr Thr Ala Ala Val Gly Ile Ser Ala Leu Ala Ala 260 265 270 Gly Ala Val Thr Tyr Ala Leu Leu Arg Arg Phe Ala Phe Gln Pro 275 280 285 46 861 DNA Mycobacterium tuberculosis 46 gtgaccgtct ccgactcgcc cgcccagcgg caaaccccac cgcaaacacc gggaggcacc 60 gctccgcgag cccgcaccgc ggcctttttc gacctggaca agaccatcat tgccaagtcc 120 agcacactgg cgttcagcaa acctttcttc gctcagggac tgctcaaccg ccgcgccgtg 180 ctgaagtcca gctacgcgca gttcatcttt ctgctgtccg gtgctgacca tgaccagatg 240 gaccggatgc gcacccacct gaccaacatg tgcgccggtt gggacgtagc ccaggtgcgg 300 tcgatagtca acgaaaccct gcacgacatc gtgaccccac tggtgttcgc cgaggccgcg 360 gacctcatcg ccgcccacaa gctgtgcggc cgcgacgtcg tggtggtctc ggcttcgggc 420 gaggagatcg tcggcccgat cgcccgcgcg ctgggcgcga cccatgcgat ggcgacccgg 480 atgatcgtcg aggacggcaa gtacacaggc gaggtcgcgt tctactgcta cggcgaaggt 540 aaggcgcaag ccatccgtga gctggctgcc agtgagggct acccgctgga acactgctac 600 gcgtactccg actcgatcac cgatctgccg atgcttgagg cggttgggca tgcctcggtg 660 gtcaaccctg atcgcggctt acgaaaggaa gccagcgtgc gcggttggcc cgtgttgtcg 720 ttctctcggc cggtgtcgct gcgcgaccgg atcccggcac cgtcagccgc ggcgatcgcc 780 acgactgcgg cggtgggtat cagcgcccta gccgccggcg cggtcaccta cgcgctacta 840 cgccgcttcg cgtttcagcc c 861 

1. A therapeutic agent for combatting mycobacterial infections, comprising an isolated M. tuberculosis peptide, or a fragment or derivative or variant thereof, wherein the peptide is encoded by an M. tuberculosis gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis wherein the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and
 45. 2. A therapeutic agent according to claim 1, wherein said M. tuberculosis gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions and a dissolved oxygen tension of less than 10% when measured at 37° C.
 3. A therapeutic agent according to claim 1 or claim 2, wherein said gene is down-regulated at a time point at least 25 days post-inoculation.
 4. A method of identifying a mycobacterial gene the expression of which is down-regulated during microbial latency, said method comprising: culturing a first mycobacterium under culture conditions that are nutrient-starving and that do not support exponential growth of the first mycobacterium; culturing a second mycobacterium under culture conditions that are not nutrient-starving and that support exponential growth of the second mycobacterium; obtaining first and second mRNA populations from said first and second mycobacteria, respectively wherein said first mRNA population is obtained from the first mycobacterium that has been harvested during stationary phase and wherein the second mRNA is obtained from the second mycobacterium that has been harvested during exponential phase growth; preparing first and second cDNA populations from said first and second mRNA populations, respectively, during which cDNA preparation a detectable label is introduced into the cDNA molecules of the first and second cDNA populations; isolating corresponding first and second cDNA molecules from the first and second cDNA populations, respectively; comparing relative amounts of label or corresponding signal emitted from the label present in the isolated first and second cDNA molecules; identifying a greater amount of label or signal provided by the isolated second cDNA molecule than that provided by the isolated first cDNA molecule; and identifying the first cDNA and the corresponding mycobacterial gene that is down-regulated during culture of a mycobacterium under nutrient-starving conditions.
 5. A method according to claim 4, wherein the corresponding first and second cDNA molecules are isolated from the first and second cDNA populations, respectively, by hybridisation thereof to an array plate containing immobilised amplified DNA sequences which have been generated from mycobacterial genomic DNA, said immobilised sequences being representative of each known gene of the mycobacterial genome, and each representative sequence having been immobilised at an identified location on the plate.
 6. A method according to claim 4 or claim 5, wherein the first mycobacterium is cultured under culture conditions defined by a dissolved oxygen tension of less than 10% air saturation when measured at 37° C., and wherein the first mycobacterium is harvested under said culture conditions.
 7. A method according to claim 4, wherein a relative down-regulation is identified by a relative 3-fold decrease in the amount of label or signal provided by the isolated first cDNA molecule over that provided by the isolated second cDNA molecule.
 8. An inhibitor of an M. tuberculosis peptide, wherein the peptide is encoded by an M. tuberculosis gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis, and wherein the inhibitor is capable of preventing or inhibiting the M. tuberculosis peptide from exerting its native biological effect wherein the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and
 45. 9. An inhibitor according to claim 8, wherein the inhibitor is capable of inhibiting a protein selected from the group consisting of: endoglucanase, endo-1,4-beta-glucanase, carboxymethyl cellulase, inorganic phosphate transporter protein, transcriptional regulatory protein, 50S ribosomal protein L3, ribosomal protein S1, 30S ribosomal protein S4, uroporphyrin III C-methyltransferase, uroporphyrinogen III methylase, urogen III methylase, crystathionine gamma synthase, O-succinylhomoserine[thiol]-lyase, and zinc metalloprotease.
 10. An inhibitor according to claim 8, selected from the group consisting of: an antibiotic capable of targeting the down-regulated M. tuberculosis gene, or the gene product thereof; and an antisense or triplex-forming nucleic acid sequence which is complementary to at least part of the down-regulated gene.
 11. An antibody that binds to a peptide encoded by a gene, or to a fragment or variant or derivative of said peptide, the expression of which gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis, wherein the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and
 45. 12. An attenuated M. tuberculosis mycobacterium in which a gene has been modified thereby rendering the mycobacterium substantially reduced in ability to enter a latent state, wherein the gene is a gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis culture and wherein said gene has a wild-type coding sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and
 46. 13. An attenuated microbial carrier, comprising a peptide encoded by a gene, or a fragment or variant or derivative of said peptide, the expression of which gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis culture, and wherein the peptide is selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, and
 45. 14. An attenuated microbial carrier according to claim 13, wherein the attenuated microbial carrier is attenuated salmonella, attenuated vaccinia virus, attenuated fowlpox virus, or attenuated M. bovis.
 15. A DNA plasmid comprising a promoter, a polyadenylation signal, and a DNA sequence that corresponds to the coding sequence of a gene or a fragment or derivative or variant of said DNA sequence, the expression of which gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis culture, wherein the promoter and polyadenylation signal are operably linked to the DNA sequence, and wherein the coding sequence is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and
 46. 16. A DNA plasmid according to claim 15, wherein the promoter is selected from the group consisting of CMV and SV40 promoters, and/or the polyadenylation signal is selected from SV40 and bovine growth hormone polyadenylation signals.
 17. An isolated RNA sequence that is encoded by a gene or a fragment or variant or derivative of said gene, wherein said gene is a gene that is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis, and wherein said gene has a coding sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and
 46. 18. An RNA vector comprising the RNA sequence of claim 17 and an integration site for a chromosome of a host cell.
 19. A medicament for treating or preventing a mycobacterial infection comprising an isolated M. tuberculosis peptide, or a fragment or derivative or variant thereof, wherein the peptide is encoded by an M. tuberculosis gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis wherein the peptide is selected from the group consisting of (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45; (ii) an inhibitor according to any of claims 8-10; (iii) an antibody according to claim 11; (iv) an attenuated M. tuberculosis mycobacterium according to claim 12; (v) an attenuated microbial carrier according to claim 13 or 14; (vi) a DNA sequence corresponding to the coding sequence of a gene or a fragment or variant or derivative of said gene which gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis, wherein the coding sequence is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46; (vii) a DNA plasmid according to claim 15 or 16; (viii) an RNA sequence according to claim 17; and (ix) an RNA vector according to claim
 18. 20. A method of treating or preventing a mycobacterial infection, comprising administering to a patient an isolated M. tuberculosis peptide, or a fragment or derivative or variant thereof, wherein the peptide is encoded by an M. tuberculosis gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis wherein the peptide is selected from the group consisting of (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45; (ii) an inhibitor according to any of claims 8-10; (iii) an antibody according to claim 11; (iv) an attenuated M. tuberculosis mycobacterium according to claim 12; (v) an attenuated microbial carrier according to any of claims 13-14; (vi) a DNA sequence corresponding to the coding sequence of a gene or a fragment or a variant or derivative of said gene which gene is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis, wherein the coding sequence is selected from the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, and 46; (vii) a DNA plasmid according to claim 15 or 16; (viii) an RNA sequence according to claim 17,; and (ix) an RNA vector according to claim
 18. 21. A diagnostic reagent for identifying an M. tuberculosis infection comprising an isolated M. tuberculosis peptide, or a fragment or derivative or variant thereof, wherein the peptide is encoded by an M. tuberculosis gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis wherein the peptide is selected from the group consisting of (i) SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, or 45; (ii) an antibody according to claim 11; and (iii) a polynucleotide probe comprising at least 8 nucleotides wherein said probe binds to at least part of a gene the expression of which is down-regulated during a stationary phase culture of M. tuberculosis under nutrient-starving culture conditions when compared with an exponential phase culture of M. tuberculosis under culture conditions that are not nutrient-starving and that support exponential growth of said M. tuberculosis.
 22. A therapeutic agent for combatting mycobacterial infections, an inhibitor, an antibody, an attenuated M. tuberculosis mycobacterium, an attenuated microbial carrier, an isolated RNA molecule, an RNA vector, or a DNA plasmid, substantially as hereinbefore described with reference to the Examples.
 23. A therapeutic agent according to claim 1 or claim 2, wherein said gene is down-regulated at a time point at least 30 days post-inoculation.
 24. A therapeutic agent according to claim 1 or claim 2, wherein said gene is down-regulated at a time point at least 40 days post-inoculation.
 25. A method according to claim 4 or claim 5, wherein the first mycobacterium is cultured under culture conditions defined by a dissolved oxygen tension of less than 7% air saturation when measured at 37° C., and wherein the first mycobacterium is harvested under said culture conditions.
 26. A method according to claim 4 or claim 5, wherein the first mycobacterium is cultured under culture conditions defined by a dissolved oxygen tension of less than 5% air saturation when measured at 37° C., and wherein the first mycobacterium is harvested under said culture conditions.
 27. An attenuated microbial carrier according to claim 14, wherein the attenuated microbial carrier is a BCG strain of attenuated M. bovis.
 28. A method according to claim 4, wherein a relative down-regulation is identified by a relative 4-fold decrease in the amount of label or signal provided by the isolated first cDNA molecule over that provided by the isolated second cDNA molecule. 